Altering the immunosuppressive microenvironment that exists within a tumor will likely be necessary for cancer vaccines to trigger an effective antitumor response. Monocyte chemoattractant proteins (such as CCL2) are produced by many tumors and have both direct and indirect immunoinhibitory effects. We hypothesized that CCL2 blockade would reduce immunosuppression and augment vaccine immunotherapy. Anti-murine CCL2/CCL12 monoclonal antibodies were administered in three immunotherapy models: one aimed at the human papillomavirus E7 antigen expressed by a non-small cell lung cancer (NSCLC) line, one targeted to mesothelin expressed by a mesothelioma cell line, and one using an adenovirus-expressing IFN-alpha to treat a nonimmunogenic NSCLC line. We evaluated the effect of the combination treatment on tumor growth and assessed the mechanism of these changes by evaluating cytotoxic T cells, immunosuppressive cells, and the tumor microenvironment. Administration of anti-CCL2/CCL12 antibodies along with the vaccines markedly augmented efficacy with enhanced reduction in tumor volume and cures of approximately half of the tumors. The combined treatment generated more total intratumoral CD8+ T cells that were more activated and more antitumor antigen-specific, as measured by tetramer evaluation. Another important potential mechanism was reduction in intratumoral T regulatory cells. CCL2 seems to be a key proximal cytokine mediating immunosuppression in tumors. Its blockade augments CD8+ T-cell immune response to tumors elicited by vaccines via multifactorial mechanisms. These observations suggest that combining CCL2 neutralization with vaccines should be considered in future immunotherapy trials.

Traumatic brain injury (TBI) affects millions of people worldwide every year. The primary impact initiates the secretion of pro- and anti-inflammatory factors, subsequent recruitment of peripheral immune cells, and activation of brain-resident microglia and astrocytes. Chemokines are major mediators of peripheral blood cell recruitment to damaged tissue, including the TBI brain. Here we review the involvement of specific chemokine pathways in TBI pathology and attempts to modulate these pathways for therapeutic purposes. We focus on chemokine (C-C motif) ligand 2/chemokine (C-C motif) receptor 2 (CCL2/CCR2) and chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 (CXCL12/CXCR4). Recent microarray and multiplex expression profiling have also implicated CXCL10 and CCL5 in TBI pathology. Chemokine (C-X3-C motif) ligand 1/chemokine (C-X3-C motif) receptor 1 (CX3CL1/CX3CR1) signaling in the context of TBI is also discussed. Current literature suggests that modulating chemokine signaling, especially CCL2/CCR2, may be beneficial in TBI treatment.

Chemokines are key mediators of the selective migration of leukocytes that occurs in neurodegenerative diseases and related inflammatory processes. Astrocytes, the most abundant cell type in the CNS, have an active role in brain inflammation. To ascertain the role of astrocytes during neuropathological processes, we have investigated in two models of primary cells (human fetal and simian adult astrocytes) the repertoire of chemokines and their receptors expressed in response to inflammatory stimuli. We demonstrated that, in the absence of any stimulation, human fetal and simian adult astrocytes express mRNA for receptors APJ, BOB/GPR15, Bonzo/CXCR6, CCR2, CCR3, CCR5, CCR8, ChemR23, CXCR3/GPR9, CXCR4, GPR1, and V28/CX3CR1. Moreover, TNFalpha and IL-1beta significantly increase BOB/GPR15, CCR2, and V28/CX3CR1 mRNA levels in both models. Furthermore, TNFalpha and IFNgamma act synergistically to induce expression of the major coreceptors for HIV infection, CXCR4 and CCR5, at both the mRNA and protein levels in human and simian astrocytes, whereas CCR3 expression was not affected by cytokine treatment. Finally, TNFalpha/IFNgamma was the most significant cytokine combination in leading to a pronounced upregulation in a comparable, time-dependent manner of the production of chemokines IP-10/CXCL10, RANTES/CCL5, MIG/CXCL9, MCP-1/CCL2, and IL-8/CXCL8. In summary, these data suggest that astrocytes serve as an important source of chemokines under the dependence of a complex cytokine regulation, and TNFalpha and IFNgamma are important modulators of chemokines and chemokine receptor expression in human as well as simian astrocytes. Finally, with the conditions we used, there was no difference between species or age of tissue.

CCR2 chemokine receptor signaling has been implicated in the generation of diverse types of neuropathology, including neuropathic pain. For example, ccr2 knock-out mice are resistant to the establishment of neuropathic pain, and mice overexpressing its ligand, monocyte chemoattractant protein-1 (MCP1; also known as CCL2), show enhanced pain sensitivity. However, whether CCR2 receptor activation occurs in the central or peripheral nervous system in states of neuropathic pain has not been clear. We developed a novel method for visualizing CCR2 receptor activation in vivo by generating bitransgenic reporter mice in which the chemokine receptor CCR2 and its ligand MCP1 were labeled by the fluorescent proteins enhanced green fluorescent protein and monomeric red fluorescent protein-1, respectively. CCR2 receptor activation under conditions such as acute inflammation and experimental autoimmune encephalomyelitis could be faithfully visualized by using these mice. We examined the status of CCR2 receptor activation in a demyelination injury model of neuropathic pain and found that MCP1-induced CCR2 receptor activation mainly occurred in the peripheral nervous system, including the injured peripheral nerve and dorsal root ganglia. These data explain the rapid antinociceptive effects of peripherally administered CCR2 antagonists under these circumstances, suggesting that CCR2 antagonists may ameliorate pain by inhibiting CCR2 receptor activation in the periphery. The method developed here for visualizing CCR2 receptor activation in vivo may be extended to G-protein-coupled receptors (GPCRs) in general and will be valuable for studying intercellular GPCR-mediated communication in vivo.

In vitro studies have implicated chemokine receptors in consumption and clearance of specific ligands. We studied the role that various signaling chemokine receptors play during ligand homeostasis in vivo. We examined the levels of ligands in serum and CNS tissue in mice lacking chemokine receptors. Compared with receptor-sufficient controls, Cx3cr1(-/-) mice exhibited augmented levels of CX3CL1 both in serum and brain, and circulating levels of CXCL1 and CXCL2 were increased in Cxcr2(-/-) mice. CCR2-deficient mice showed significantly increased amounts of circulating CCL2 compared with wild-type mice. Cxcr3(-/-) mice revealed increased levels of circulating and brain CXCL10 after experimental autoimmune encephalomyelitis (EAE) induction. CCR2-deficient peripheral blood and resident peritoneal cells exhibited reduced binding capacity and biologic responses to the CCR1 ligand CCL3, suggesting that elevated levels of CCR2 ligands had down-regulated CCR1. The results indicate that signaling chemokine receptors clear chemokines from circulation and tissues. These homeostatic functions of signaling chemokine receptors need to be integrated into safety and efficacy calculations when considering therapeutic receptor blockade.

Microarray analysis of human alcoholic brain and cultured cells exposed to ethanol showed significant changes in expression of genes related to immune or inflammatory responses, including chemokines and chemokine receptors. To test the hypothesis that chemokines exhibit previously undiscovered pleiotropic effects important for the behavioral actions of ethanol, we studied mutant mice with deletion of the Ccr2, Ccr5, Ccl2 or Ccl3 genes. Deletion of Ccr2, Ccl2 (females) or Ccl3 in mice resulted in lower preference for alcohol and consumption of lower amounts of alcohol in a two-bottle choice test as compared with wild-type mice. Ethanol treatment (2.5 g/kg, i.p.) induced stronger conditioned taste aversion in Ccr2, Ccl2 or Ccl3 null mutant mice than in controls. Ccr2 and Ccr5 null mutant mice did not differ from wild-type mice in ethanol-induced loss of righting reflex (LORR), but mice lacking Ccl2 or Ccl3 showed longer LORR than wild-type mice. There were no differences between mutant strains and wild-type mice in severity of ethanol-induced withdrawal. Genetic mapping of chromosome 11 for the Ccl2 and Ccl3 genes (46.5 and 47.6 cM, respectively) revealed that an alcohol-induced LORR QTL region was contained within the introgressed region derived from 129/SvJ, which may cause some behavioral phenotypes observed in the null mice. On the contrary, known QTLs on Chr 9 are outside of 129/SvJ region in Ccr2 and Ccr5 (71.9 and 72.0 cM, respectively) null mutant mice. These data show that disruption of the chemokine network interferes with motivational effects of alcohol.

Macrophage infiltration is implicated in various types of pulmonary fibrosis. One important pathogenetic process associated with pulmonary fibrosis is injury to basement membranes by matrix metalloproteinases (MMPs) that are produced mainly by macrophages. In this study, C-C chemokine receptor 2-deficient (CCR2-/-) mice were used to explore the relationship between macrophage infiltration and MMP activity in the pathogenesis of pulmonary fibrosis, using the bleomycin-induced model of this disease process. CCR2 is the main (if not only) receptor for monocyte chemoattractant protein-1/C-C chemokine ligand 2 (MCP-1/CCL2), which is a critical mediator of macrophage trafficking, and CCR2 -/- mice demonstrate defective macrophage migration. Pulmonary fibrosis was induced in CCR2-/- and wild-type (CCR2+/+) mice by intratracheal instillation of bleomycin. No significant differences in the total protein concentration in bronchoalveolar lavage (BAL) fluid, or in the degree of histological lung inflammation, were observed in the two groups until day 7. Between days 3 and 21, however, BAL fluid from CCR2-/- mice contained fewer macrophages than BAL fluid from CCR2+/+ mice. Gelatin zymography of BAL fluid and in situ zymography revealed reduced gelatinolytic activity in CCR2-/- mice. Immunocytochemical staining showed weaker expression of MMP-2 and MMP-9 in macrophages in BAL fluid from CCR2-/- mice at day 3. Gelatin zymography of protein extracted from alveolar macrophages showed reduced gelatinolytic activity of MMP-2 and MMP-9 in CCR2-/- mice. At days 14 and 21, lung remodelling and the hydroxyproline content of lung tissues were significantly reduced in CCR2-/- mice. These results suggest that the CCL2/CCR2 functional pathway is involved in the pathogenesis of bleomycin-induced pulmonary fibrosis and that CCR2 deficiency may improve the outcome of this disease by regulating macrophage infiltration and macrophage-derived MMP-2 and MMP-9 production.

Neuroinflammation is a prominent feature of many neurodegenerative diseases, however, little is known about neuroinflammation in Huntington's disease. We used quantitative real time-PCR to compare the expression level of neuroinflammation-associated mediators in the striatum, cortex, and cerebellum from post-mortem Huntington's disease patient samples with controls. We found increased expression of several key inflammatory mediators, including CCL2 and IL-10, specifically in the striatum of Huntington's disease patients, the main area affected by this pathology. Remarkably, we also found upregulation of IL-6, IL-8, and MMP9, in the cortex and notably the cerebellum, a brain area commonly thought to be spared by Huntington's disease. Our data suggest that neuroinflammation is a prominent feature associated with Huntington's disease and may constitute a novel target for therapeutic intervention.

Half of patients with muscle-invasive bladder cancer develop metastatic disease, and this is responsible for most of the deaths from this cancer. Low expression of RhoGTP dissociation inhibitor 2 (RhoGDI2; also known as ARHGDIB and Ly-GDI) is associated with metastatic disease in patients with muscle-invasive bladder cancer. Moreover, a reduction in metastasis is observed upon reexpression of RhoGDI2 in xenograft models of metastatic cancer. Here, we show that RhoGDI2 suppresses lung metastasis in mouse models by reducing the expression of isoforms V1 and V3 of the proteoglycan versican (VCAN; also known as chondroitin sulfate proteoglycan 2 [CSPG2]). In addition, we found that high versican levels portended poor prognosis in patients with bladder cancer. The functional importance of tumor expression of versican in promoting metastasis was established in in vitro and in vivo studies in mice that implicated a role for the chemokine CCL2 (also known as MCP1) and macrophages. Further analysis indicated that RhoGDI2 suppressed metastasis by altering inflammation in the tumor microenvironment. In summary, we demonstrate what we believe to be a new mechanism of metastasis suppression that works by reducing host responses that promote metastatic colonization of the lung. Therapeutic targeting of these interactions may provide a novel adjuvant strategy for delaying the appearance of clinical metastasis in patients.

Thymic stromal lymphopoietin (TSLP) is highly expressed by bronchial epithelial cells and skin keratinocytes in allergic diseases. TSLP acts as a master switch for allergic inflammation through the activation of dendritic cells and mast cells for initiating inflammatory type 2 T-helper lymphocyte responses. To elucidate the immunological cascades of epithelium/keratinocyte-eosinophil-mediated allergic inflammation, we examined the modulating effects of TSLP on human eosinophils. Expression of TSLP receptor complex was detected by RT-PCR, flow cytometry, and Western blot. Adhesion molecules, cytokine, and chemokines were quantitated by flow cytometry or ELISA. Intracellular signal transduction molecules were measured by Western blot and flow cytometry. We observed that human eosinophils constitutively expressed functional heterodimeric TSLP receptor complex comprising TSLP-binding chain TSLPR and IL-7Ralpha chain. TSLP could significantly delay eosinophil apoptosis, up-regulate cell surface expression of adhesion molecule CD18 and intercellular adhesion molecule-1, but down-regulate L-selectin, enhance eosinophil adhesion onto fibronectin, and induce the release of inflammatory cytokine IL-6 and chemokines CXCL8, CXCL1, and CCL2 (all P < 0.05). All these effects were concentration dependent and TSLP specific. TSLP regulated the above effects through the activation of extracellular signal-regulated protein kinase, p38 mitogen-activated protein kinase, and NF-kappaB signaling pathway, but not signal transducer and activator of transcription 5 and 3, which were usually activated in other effector cells upon TSLP stimulation. Collectively, the above findings elucidate the proallergic mechanisms of TSLP via the activation of distinct intracellular signaling pathways in eosinophils.

OBJECTIVE

Leukotriene B4 (LTB4), a potent leukocyte chemoattractant, is known to promote several inflammatory diseases, including atherosclerosis. We sought to determine mechanisms through which LTB4 modulates atherosclerosis in cell lines expressing LTB4 receptors, BLT-1, and in mice deficient in BLT-1 as well as macrophage cell lines derived from BLT-1+/+ and BLT-1-/- mice.

RESULTS

Analysis of global changes in gene expression induced by LTB4 in rat basophilic leukemia cells (RBL-2H3) expressing the human BLT-1 showed highest-fold increase in expression of fatty acid translocase/CD36 and the chemokine MCP1/JE/CCL2, which are critical in atherogenesis. To determine the importance of BLT-1 in atherogenesis, we crossed BLT-1-null mice with apolipoprotein (apo)-E-deficient mice, which develop severe atherosclerosis. Deletion of BLT-1 significantly reduced the lesion formation in apo-E-/- mice only during initiating stages (4 and 8 weeks) but had no effect on the lesion size in mice fed atherogenic diet for 19 weeks. Macrophage cell lines from BLT-1-deficient mice expressed the low-affinity LTB4 receptor, BLT-2, and exhibited chemotaxis to LTB4.

CONCLUSIONS

The effects of LTB4 in atherosclerosis are likely mediated through the high-affinity BLT-1 and the low-affinity BLT-2 receptors. LTB4 promotes atherosclerosis by chemo-attracting monocytes, by providing an amplification loop of monocyte chemotaxis via CCL2 production, and by converting monocytes to foam cells by enhanced expression of CD36 and fatty acid accumulation.

BACKGROUND

Inflammation and matrix degradation are the hallmarks of high-risk atherosclerosis that leads to myocardial infarction and stroke. Toll-like receptors (TLRs), key players in innate immunity, are upregulated in atherosclerotic lesions, but their functional role in human atherosclerosis is unknown. We explored the effects of blocking TLR-2, TLR-4, and myeloid differentiation primary response gene 88 (MyD88), a signaling adaptor shared by most TLRs and interleukin-1 receptor (IL-1R), in an in vitro model of human atherosclerosis.

RESULTS

Carotid endarterectomies were obtained from patients with symptomatic carotid disease. Cells were isolated via enzymatic tissue dissociation and cultured in the presence or absence of TLR signaling blockers. A dominant-negative form of MyD88 (MyD88(DN)) decreased the production of monocyte chemotactic protein-1/CCL2 (P=0.000), IL-8/CXCL8 (P=0.006), IL-6 (P=0.002), matrix metalloproteinase-1 (MMP-1; P=0.002), and MMP-3 (P=0.000), as well as nuclear factor-kappaB activation (P<0.05) in atheroma cell cultures. IL-1R antagonist, TLR-4 blocking antibodies, or overexpression of a dominant-negative form of the TLR-4 signaling adaptor TRIF-related adaptor molecule reduced nuclear factor-kappaB activity but did not have a broad impact on the production of the mediators studied. In contrast, TLR-2 neutralizing antibodies inhibited nuclear factor-kappaB activation (P<0.05) and significantly reduced monocyte chemotactic protein-1/CCL2 (P=0.000), IL-8/CXCL8 (P=0.009), IL-6 (P=0.000), and MMP-1 (P=0.000), MMP-2 (P=0.004), MMP-3 (P=0.000), and MMP-9 (P=0.006) production.

CONCLUSIONS

Our data indicate that TLR-2 signaling through MyD88 plays a predominant role in inflammation and matrix degradation in human atherosclerosis. TLR-2 blockade may represent a therapeutic strategy for atherosclerosis and its complications.

Tumor-infiltrating lymphocytes (TILs) are important prognostic factors in cancer progression and key players in cancer immunotherapy. Although γδ T lymphocytes can target a diversity of tumor cell types, their clinical manipulation is hampered by our limited knowledge of the molecular cues that determine γδ T cell migration toward tumors in vivo. In this study we set out to identify the chemotactic signals that orchestrate tumor infiltration by γδ T cells. We have used the preclinical transplantable B16 melanoma model to profile chemokines in tumor lesions and assess their impact on γδ TIL recruitment in vivo. We show that the inflammatory chemokine CCL2 and its receptor CCR2 are necessary for the accumulation of γδ TILs in B16 lesions, where they produce IFN-γ and display potent cytotoxic functions. Moreover, CCL2 directed γδ T cell migration in vitro toward tumor extracts, which was abrogated by anti-CCL2 neutralizing Abs. Strikingly, the lack of γδ TILs in TCRδ-deficient but also in CCR2-deficient mice enhanced tumor growth in vivo, thus revealing an unanticipated protective role for CCR2/CCL2 through the recruitment of γδ T cells. Importantly, we demonstrate that human Vδ1 T cells, but not their Vδ2 counterparts, express CCR2 and migrate to CCL2, whose expression is strongly deregulated in multiple human tumors of diverse origin, such as lung, prostate, liver, or breast cancer. This work identifies a novel protective role for CCL2/CCR2 in the tumor microenvironment, while opening new perspectives for modulation of human Vδ1 T cells in cancer immunotherapy.

Chemokines are important mediators in immune responses and inflammatory processes. Calcitonin gene-related peptide (CGRP) is produced in dorsal root ganglion (DRG) neurons. In this study, CGRP radioimmunoassay was used to investigate whether the chemokines CCL2 and CXCL1 could trigger CGRP release from cultured DRG neurons of neonatal rats and, if so, which cellular signaling pathway was involved. The results showed that CCL2 and CXCL1 ( approximately 5-100 ng/ml) evoked CGRP release and intracellular calcium elevation in a pertussis toxin (PTX)-sensitive manner. The CGRP release by CCL2 and CXCL1 was significantly inhibited by EGTA, omega-conotoxin GVIA (an N-type calcium channel blocker), thapsigargin, and ryanodine. Pretreatment of DRG neurons for 30 min with the inhibitors of phospholipase C (PLC) and protein kinase C (PKC) but not mitogen-activated protein kinases (MAPKs) significantly reduced CCL2- or CXCL1-induced CGRP release and intracellular calcium elevation. Intraplantar injection of CCL2 or CXCL1 produced hyperalgesia to thermal and mechanical stimulation in rats. These data suggest that CCL2 and CXCL1 can stimulate CGRP release and intracellular calcium elevation in DRG neurons. PLC-, PKC-, and calcium-induced calcium release from ryanodine-sensitive calcium stores signaling pathways are involved in CCL2- and CXCL1-induced CGRP release from primary nociceptive neurons, in which chemokines produce painful effects via direct actions on chemokine receptors expressed by nociceptive neurons.

Astrocytes play key roles in CNS development, inflammation, and repair by producing a wide variety of cytokines, chemokines, and growth factors. Understanding the regulation of this network is important for a full understanding of astrocyte functioning. In this study, expression levels of 268 genes encoding cytokines, chemokines, growth factors, and their receptors were established in cultured human adult astrocytes using cDNA arrays. Also, changes in this gene profile were determined following stimulation with TNFalpha, IL-1beta, and IFNgamma. The data obtained reveal a highly reproducible pattern of gene expression not only between different astrocyte cultures from a single source, but also between astrocytes from different donors. They also identify several gene products not previously described for human astrocytes, including a.o. IL-17, CD70, CD147, and BIGH3. When stimulated with TNFalpha astrocytes respond with increased expression of several genes, notably including those encoding the chemokines CCL2 (MCP-1), CCL5 (RANTES), and CXCL8 (IL-8), growth factors including BMP-2A, BMP-3, neuromodulin (GAP43), BDNF, and G-CSF, and receptors such as the CRF receptor, the calcitonin receptor (CTR), and TKT. The response to IL-1beta involves largely the same range of genes, but responses were blunted in comparison to the TNFalpha response. Treatment with IFNgamma had no or only marginal effects on expression of any of the 268 genes analyzed. Astrocytes treated with a mixture of all three stimuli together displayed responses that are largely similar to those found in response to TNFalpha or IL-1beta alone, with only few additional synergistic effects.

Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness worldwide in the elderly population. The pathology of dry AMD consists of macular degeneration of photoreceptors and the RPE, lipofuscin (A2E) accumulation, and drusen formation. Mice have been widely used for generating models that simulate human AMD features for investigating the pathogenesis, treatment and prevention of the disease. Although the mouse has no macula, focal atrophy of photoreceptors and RPE, lipofuscin accumulation, and increased A2E can develop in aged mouse eyes. However, drusen are rarely seen in mice because of their simpler Bruch's membrane and different process of lipofuscin extrusion compared with humans. Thus, analyzing basal deposits at the ultrastructural level and understanding the ultrastructural pathologic differences between various mouse AMD models are critical to comprehending the significance of research findings and response to possible therapeutic options for dry AMD. Based on the multifactorial pathogenesis of AMD, murine dry AMD models can be classified into three groups. First, genetically engineered mice that target genes related to juvenile macular dystrophies are the most common models, and they include abcr(-/-) (Stargardt disease), transgenic ELOVL4 (Stargardt-3 dominant inheritary disease), Efemp1(R345W/R345W) (Doyne honeycomb retinal dystrophy), and Timp3(S156C/S156C) (Sorsby fundus dystrophy) mice. Other murine models target genes relevant to AMD, including inflammatory genes such as Cfh(-/-), Ccl2(-/-), Ccr2(-/-), Cx3cr1(-/-), and Ccl2(-/-)/cx3cr1(-/-), oxidative stress associated genes such as Sod1(-/-) and Sod2 knockdown, metabolic pathway genes such as neprilysin(-/-) (amyloid beta), transgenic mcd/mcd (cathepsin D), Cp(-/-)/Heph(-/Y) (ferroxidase ceruloplasmin/hepaestin, iron metabolism), and transgenic ApoE4 on high fat and high cholesterol diet (lipid metabolism). Second, mice have also been immunologically manipulated by immunization with carboxyethylpyrrole (CEP), an oxidative fragment of DHA found in drusen, and found to present with dry AMD features. Third, natural mouse strains such as arrd2/arrd2 (Mdm gene mutation) and the senescence accelerated mice (SAM) spontaneously develop features of dry AMD like photoreceptor atrophy and thickening of Bruch's membrane. All the aforementioned models develop retinal lesions with various features that simulate dry AMD lesions: focal photoreceptor degeneration, abnormal RPE with increased lipofuscin, basal infolding, decreased melanosomes and degeneration. However, Bruch's membrane changes are less common. Most mice develop retinal lesions at an older age (6-24 months, depending on the models), while the Ccl2(-/-)/cx3cr1(-/-) mice develop lesions by 4-6 weeks. Although murine models present various degrees of retinal and/or RPE degeneration, classical drusen is extremely rare. Using electron microscopy, small drusenoid deposits are found between RPE and Bruch's membrane in a few models including Efemp1(R345W/R345W), Ccl2(-/-)/cx3cr1(-/-), neprilysin(-/-), transgenic mcd/mcd, and ApoE4 transgenic mice on a high fat diet. High A2E levels are measured in the retinas of abcr(-/-), transgenic ELOVL4, and Ccl2(-/-)/cx3cr1(-/-) mice. In summary, murine models provide useful tools for studying AMD pathogenesis and evaluating novel therapies for this disease. This review compares the major dry AMD murine models and discusses retinal pathology at the ultrastructural level.

OBJECTIVE

To extensively characterize the complex network of cytokines present in uveitis aqueous humor (AqH), and the relationships between cytokines and the cellular infiltrate.

METHODS

AqH from noninflammatory control subjects and patients with idiopathic, Fuchs' heterochromic cyclitis (FHC), and herpes-viral or Behçet's uveitis were analyzed for IL-1beta, -2, -4, -5, -7, -8, -10, -12, -13, -15, TNFalpha, IFNgamma, CCL2 (MCP-1), CCL5 (RANTES), CCL11 (Eotaxin), TGFbeta2, and CXCL12 (SDF-1), using multiplex bead immunoassays. The cellular infiltrate was also determined for each sample.

RESULTS

Idiopathic uveitis AqH, compared with noninflammatory controls, was characterized by high levels of IL-6, IL-8, CCL2 and IFNgamma, the levels of which correlated with each other. For IL-6 and IL-8 these levels were proportional to the number of neutrophils present. By contrast, the levels of both TGFbeta2 and CXCL12 decreased in idiopathic uveitis AqH with increasing inflammation. Cluster analysis showed a degree of segregation between noninflammatory and idiopathic uveitis AqH. Further examination using random forest analysis yielded a complete distinction between these two groups. The minimum cytokines required for this classification were IL-6, IL-8, CCL2, IL-13, TNFalpha, and IL-2.

CONCLUSIONS

Application of multiplex bead immunoassays has allowed us to identify distinct patterns of cytokines that relate to both clinical disease and the cellular infiltrates present. Bioinformatics analysis allowed identification of cytokines that differentiate idiopathic uveitis from noninflammatory control AqH and are likely to be important for the pathogenesis of uveitis.

The infiltration of leukocytes plays a major role in mediating tubulointerstitial inflammation and fibrosis in chronic renal disease. CC chemokines participate in leukocyte migration and infiltration into inflamed renal tissue. Because CC chemokine-directed leukocyte migration is mediated by target cell expression of a group of CC chemokine receptors, this study examined the expression of CC chemokines and their receptors during initiation of tubulointerstitial fibrosis after unilateral ureteral obstruction in C57BL/6 mice. Obstructed kidneys developed hydronephrosis, tubular cell damage, interstitial inflammation, and fibrosis. From days 2 to 10, a progressive interstitial influx of F4/80+ macrophages and CD3+ lymphocytes occurred (macrophages, 4-fold; lymphocytes, 20-fold at day 10, compared with contralateral control kidneys). In parallel, the number of activated fibroblast-specific protein 1+ fibroblasts and interstitial collagen IV accumulation increased from days 2 to 10. The mRNA expression of CC chemokines (predominantly monocyte chemoattractant protein-1 [MCP-1]/CCL2, RANTES/CCL5) and their receptors CCR1, CCR2, CCR5 increased progressively from days 2 to 10. By in situ hybridization, a prominent interstitial mRNA expression of MCP-1 and RANTES and their receptors CCR2 and CCR5 localized to interstitial mononuclear cell infiltrates. MCP-1 and RANTES expression was also seen in tubular epithelial cells. Fluorescence-activated cell sorter analysis of single-cell suspensions from obstructed kidneys revealed a prominent expression of CCR2 and CCR5 by infiltrating macrophages, whereas most lymphocytes expressed CCR5 only. These data demonstrate an increased expression of MCP-1/CCL2 and RANTES/CCL5 at sites of tubulointerstitial damage and progressive fibrosis during unilateral ureteral obstruction that correlates with simultaneous accumulation of interstitial macrophages and T lymphocytes expressing the respective surface receptors CCR2 and CCR5. The chemokine receptor-mediated leukocyte influx into the tubulointerstitium could offer a new potential target for therapeutic intervention in progressive renal tubulointerstitial fibrosis.

BACKGROUND

Inflammatory cytokines may affect pulmonary vascular remodeling in idiopathic pulmonary arterial hypertension (IPAH). CC chemokine ligand 2 (CCL2) is synthesized by vascular cells and can stimulate monocyte/macrophage migration and smooth muscle cell (SMC) proliferation.

OBJECTIVE

To investigate the role of CCL2 in IPAH.

METHODS

CCL2 levels in plasma, monocytes, lungs, and medium from pulmonary endothelial cell (P-EC) or pulmonary artery SMC (PA-SMC) cultures were measured by ELISA and Western blot analysis. CCL2 receptor CCR2 mRNA levels in monocytes, P-ECs, and PA-SMCs were measured by real-time polymerase chain reaction. Effect of CCL2 on PA-SMC proliferation and migration was assessed using [3H]thymidine incorporation and a modified Boyden's chamber. The effect of endothelial cell-derived CCL2 on monocyte migration was measured using a modified Boyden's chamber.

RESULTS

Compared with control subjects, we found the following in patients with IPAH: elevated CCL2 protein levels in plasma and lung tissue, whereas monocyte CCL2 levels were similar between patients and control subjects, and elevated CCL2 release by P-ECs or PA-SMCs. P-ECs released twice as much CCL2 than did PA-SMCs. Monocyte migration was markedly increased in the presence of P-ECs, and the increase was larger with P-ECs from patients with IPAH. CCL2-blocking antibodies reduced P-ECs' chemotactic activity by 60%. Compared with controls, PA-SMCs from patients exhibited stronger migratory and proliferative responses to CCL2, in keeping with the finding that CCR2 was markedly increased in PA-SMCs from patients.

CONCLUSIONS

These results suggest that CCL2 overproduction may be a feature of the abnormal P-EC phenotype in IPAH, contributing to the inflammatory process and to pulmonary vascular remodeling.

Renal cell carcinoma (RCC), the most common human kidney cancer, is frequently infiltrated with tumor-associated macrophages (TAM) that can promote malignant progression. Here, we show that TAMs isolated from human RCC produce substantial amounts of the proinflammatory chemokine CCL2 and immunosuppressive cytokine IL-10, in addition to enhanced eicosanoid production via an activated 15-lipoxygenase-2 (15-LOX2) pathway. TAMs isolated from RCC tumors had a high 15-LOX2 expression and secreted substantial amounts of 15(S)-hydroxyeicosatetraenoic acid, its major bioactive lipid product. Inhibition of lipoxygenase activity significantly reduced production of CCL2 and IL-10 by RCC TAMs. In addition, TAMs isolated from RCC were capable of inducing in T lymphocytes, the pivotal T regulatory cell transcription factor forkhead box P3 (FOXP3), and the inhibitory cytotoxic T-lymphocyte antigen 4 (CTLA-4) coreceptor. However, this TAM-mediated induction of FOXP3 and CTLA-4 in T cells was independent of lipoxygenase and could not be reversed by inhibiting lipoxygenase activity. Collectively, our results show that TAMs, often present in RCCs, display enhanced 15-LOX2 activity that contributes to RCC-related inflammation, immunosuppression, and malignant progression. Furthermore, we show that TAMs mediate the development of immune tolerance through both 15-LOX2-dependent and 15-LOX2-independent pathways. We propose that manipulating LOX-dependent arachidonic acid metabolism in the tumor microenvironment could offer new strategies to block cancer-related inflammation and immune escape in patients with RCC.

Studies of the 1918 H1N1 influenza pandemic, the H5N1 avian influenza outbreak, and the 2009 H1N1 pandemic illustrate that sex and pregnancy contribute to severe outcome from infection, suggesting a role for sex steroids. To test the hypothesis that the sexes respond differently to influenza, the pathogenesis of influenza A virus infection was investigated in adult male and female C57BL/6 mice. Influenza infection reduced reproductive function in females and resulted in greater body mass loss, hypothermia, and mortality in females than males. Whereas lung virus titers were similar between the sexes, females had higher induction of proinflammatory cytokines and chemokines, including TNF-α, IFN-γ, IL-6, and CCL2, in their lungs than males. Removal of the gonads in both sexes eliminated the sex difference in influenza pathogenesis. Manipulation of testosterone or dihydrotestosterone concentrations in males did not significantly impact virus pathogenesis. Conversely, females administered high doses of estradiol had a ≥10-fold lower induction of TNF-α and CCL2 in the lungs and increased rates of survival as compared with females that had either low or no estradiol. The protective effects of estradiol on proinflammatory cytokines and chemokines, morbidity, and mortality were primarily mediated by signaling through estrogen receptor α (ERα). In summary, females suffer a worse outcome from influenza A virus infection than males, which can be reversed by administration of high doses of estradiol to females and reflects differences in the induction of proinflammatory responses and not in virus load.

Chronic inflammation is an underlying factor linking obesity with insulin resistance. Diet-induced obesity promotes an increase in circulating levels of inflammatory monocytes and their infiltration into expanding adipose tissue. Nevertheless, the endogenous pathways that trigger and sustain chronic low-grade inflammation in obesity are incompletely understood. In this study, we report that a high-fat diet selectively increases the circulating levels of CD11b(+) monocytes in wild-type mice that express leukotriene B(4) receptor, BLT-1, and that this increase is abolished in BLT-1-null mice. The accumulation of classically activated (M1) adipose tissue macrophages (ATMs) and the expression of proinflammatory cytokines and chemokines (i.e., IL-6 and Ccl2) was largely blunted in adipose tissue of obese BLT-1(-/-) mice, whereas the ratio of alternatively activated (M2) ATMs to M1 ATMs was increased. Obese BLT-1(-/-) mice were protected from systemic glucose and insulin intolerance and this was associated with a decrease in inflammation in adipose tissue and liver and a decrease in hepatic triglyceride accumulation. Deletion of BLT-1 prevented high fat-induced loss of insulin signaling in liver and skeletal muscle. These observations elucidate a novel role of chemoattractant receptor, BLT-1, in promoting monocyte trafficking to adipose tissue and promoting chronic inflammation in obesity and could lead to the identification of new therapeutic targets for treating insulin resistance in obesity.

The natural interferon (IFN)-producing cell is now known to be identical to the plasmacytoid dendritic cell (PDC). These are Lin-, CD123+, CD11c-, and human leukocyte antigen-DR+ cells that secrete large amounts of IFN-alpha (1-2 IU/cell) when stimulated by enveloped viruses such as herpes simplex virus. In the current study, we have evaluated chemokine expression by virally stimulated PDC. Up-regulation of mRNA for CCL4, CCL3, CCL5, CCL2, and CXC chemokine ligand (CXCL)10 in herpes simplex virus-stimulated PDC was detected by RNAse protection assays. In contrast, PDC-depleted peripheral blood mononuclear cells did not up-regulate these mRNA species upon viral stimulation. Enzyme-linked immunosorbent assay and/or intracellular flow cytometry confirmed production of these proteins, and studies indicated overlapping production of IFN-alpha and the other cytokines/chemokines by PDC. Endocytosis plays a critical role in chemokine induction, as disruption of the pathway inhibits the response. However, transcription of viral genes is not required for chemokine induction. Autocrine IFN-alpha signaling in the PDC could account for a portion of the CXCL10 and CCL2 production in virally stimulated PDC but was not responsible for the induction of the other chemokines. To evaluate the functional role of the chemokines, chemotaxis assays were performed using supernatants from virally stimulated PDC. Activated T cells and natural killer cells, but not naïve T cells, were preferentially recruited by these PDC supernatants. Migration was subsequently inhibited by addition of neutralizing antibody to CCL4 and CXCL10. We hypothesize that virally induced chemokine production plays a pivotal role in the homing of leukocytes to PDC.

BACKGROUND

Monocyte chemoattractant protein (CCL2/MCP-1) is a chemokine that attracts cells involved in the immune/inflammatory response. As microglia are one of the main cell types sustaining inflammation in brain, we proposed here to analyze the direct effects of MCP-1 on cultured primary microglia.

METHODS

Primary microglia and neuronal cultures were obtained from neonatal and embryonic Wistar rats, respectively. Microglia were incubated with different concentrations of recombinant MCP-1 and LPS. Cell proliferation was quantified by measuring incorporation of bromodeoxyuridine (BrdU). Nitrite accumulation was measured using the Griess assay. The expression and synthesis of different proteins was measured by RT-PCR and ELISA. Cell death was quantified by measuring release of LDH into the culture medium.

RESULTS

MCP-1 treatment (50 ng/ml, 24 h) did not induce morphological changes in microglial cultures. Protein and mRNA levels of different cytokines were measured, showing that MCP-1 was not able to induce proinflammatory cytokines (IL-1β, IL6, MIP-1α), either by itself or in combination with LPS. A similar lack of effect was observed when measuring inducible nitric oxide synthase (NOS2) expression or accumulation of nitrites in the culture media as a different indicator of microglial activation. MCP-1 was also unable to alter the expression of different trophic factors that were reduced by LPS treatment. In order to explore the possible release of other products by microglia and their potential neurotoxicity, neurons were co-cultured with microglia: no death of neurons could be detected when treated with MCP-1. However, the presence of MCP-1 induced proliferation of microglia, an effect opposite to that observed with LPS.

CONCLUSIONS

These data indicate that, while causing migration and proliferation of microglia, MCP-1 does not appear to directly activate an inflammatory response in this cell type, and therefore, other factors may be necessary to cause the changes that result in the neuronal damage commonly observed in situations where MCP-1 levels are elevated.