Primary tumors have been shown to prepare distal organs for later colonization of metastatic cells by stimulating organ-specific infiltration of bone marrow derived cells. Here we demonstrate that neutrophils accumulate in the lung prior to the arrival of metastatic cells in mouse models of breast cancer. Tumor-entrained neutrophils (TENs) inhibit metastatic seeding in the lungs by generating H(2)O(2) and tumor secreted CCL2 is a critical mediator of optimal antimetastatic entrainment of G-CSF-stimulated neutrophils. TENs are present in the peripheral blood of breast cancer patients prior to surgical resection but not in healthy individuals. Thus, whereas tumor-secreted factors contribute to tumor progression at the primary site, they concomitantly induce a neutrophil-mediated inhibitory process at the metastatic site.

GM-CSF and M-CSF (CSF-1) can enhance macrophage lineage numbers as well as modulate their differentiation and function. Of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. What the critical actions are of these CSFs on macrophages during inflammatory reactions are unknown. To address this issue, adherent macrophages (GM-BMM and BMM) were first derived from murine bone marrow precursors by GM-CSF and M-CSF, respectively, and stimulated in vitro with LPS to measure secreted cytokine production, as well as NF-kappaB and AP-1 activities. GM-BMM preferentially produced TNF-alpha, IL-6, IL-12p70, and IL-23 whereas, conversely, BMM generated more IL-10 and CCL2; strikingly the latter population could not produce detectable IL-12p70 and IL-23. Following LPS stimulation, GM-BMM displayed rapid IkappaBalpha degradation, RelA nuclear translocation, and NF-kappaB DNA binding relative to BMM, as well as a faster and enhanced AP-1 activation. Each macrophage population was also pretreated with the other CSF before LPS stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and NF-kappaB activity. Thus, GM-CSF and M-CSF demonstrate, at the level of macrophage cytokine production, different and even competing responses with implications for their respective roles in inflammation, including a possible dampening or suppressive role for M-CSF in certain circumstances.

Bone marrow-derived cells are known to play important roles in repair/regeneration of injured tissues, but their roles in pathological fibrosis are less clear. Here, we report a critical role for the chemokine receptor CCR2 in the recruitment and activation of lung fibrocytes (CD45(+), CD13(+), collagen 1(+), CD34(-)). Lung fibrocytes were isolated in significantly greater numbers from airspaces of fluorescein isothiocyanate-injured CCR2(+/+) mice than from CCR2(-/-) mice. Transplant of CCR2(+/+) bone marrow into CCR2(-/-) recipients restored recruitment of lung fibrocytes and susceptibility to fibrosis. Ex vivo PKH-26-labeled CCR2(+/+) lung fibrocytes also migrated to injured airspaces of CCR2(-/-) recipients in vivo. Isolated lung fibrocytes expressed CCR2 and migrated to CCL2, and CCL2 stimulated collagen secretion by lung fibrocytes. Fibrocytes could transition into fibroblasts in vitro, and this transition was associated with loss of CCR2 expression and enhanced production of collagen 1. This is the first report describing expression of CCR2 on lung fibrocytes and demonstrating that CCR2 regulates both recruitment and activation of these cells after respiratory injury.

In the central nervous system (CNS), the transcription factor nuclear factor (NF)-kappaB is a key regulator of inflammation and secondary injury processes. After trauma or disease, the expression of NF-kappaB-dependent genes is highly activated, leading to both protective and detrimental effects on CNS recovery. We demonstrate that selective inactivation of astroglial NF-kappaB in transgenic mice expressing a dominant negative (dn) form of the inhibitor of kappaB alpha under the control of an astrocyte-specific promoter (glial fibrillary acidic protein [GFAP]-dn mice) leads to a dramatic improvement in functional recovery 8 wk after contusive spinal cord injury (SCI). Histologically, GFAP mice exhibit reduced lesion volume and substantially increased white matter preservation. In parallel, they show reduced expression of proinflammatory chemokines and cytokines, such as CXCL10, CCL2, and transforming growth factor-beta2, and of chondroitin sulfate proteoglycans participating in the formation of the glial scar. We conclude that selective inhibition of NF-kappaB signaling in astrocytes results in protective effects after SCI and propose the NF-kappaB pathway as a possible new target for the development of therapeutic strategies for the treatment of SCI.

In inflammatory diseases occurring outside the CNS, communication between the periphery and the brain via humoral and/or neural routes results in central neural changes and associated behavioral alterations. We have recently identified another immune-to-CNS communication pathway in the setting of organ-centered peripheral inflammation: namely, the entrance of immune cells into the brain. In our current study, using a mouse model of inflammatory liver injury, we have confirmed the significant infiltration of activated monocytes into the brain in mice with hepatic inflammation and have defined the mechanism that mediates this trafficking of monocytes. Specifically, we show that in the presence of hepatic inflammation, mice demonstrate elevated cerebral monocyte chemoattractant protein (MCP)-1 levels, as well as increased numbers of circulating CCR2-expressing monocytes. Cerebral recruitment of monocytes was abolished in inflamed mice that lacked MCP-1/CCL2 or CCR2. Furthermore, in mice with hepatic inflammation, microglia were activated and produced MCP-1/CCL2 before cerebral monocyte infiltration. Moreover, peripheral tumor necrosis factor (TNF)alpha signaling was required to stimulate microglia to produce MCP-1/CCL2. TNFalpha signaling via TNF receptor 1 (TNFR1) is required for these observed effects since in TNFR1 deficient mice with hepatic inflammation, microglial expression of MCP-1/CCL2 and cerebral monocyte recruitment were both markedly inhibited, whereas there was no inhibition in TNFR2 deficient mice. Our results identify the existence of a novel immune-to-CNS communication pathway occurring in the setting of peripheral organ-centered inflammation which may have specific implications for the development of alterations in cerebral neurotransmission commonly encountered in numerous inflammatory diseases occurring outside the CNS.

Tumor-promoted constraints negatively affect cytotoxic T lymphocyte (CTL) trafficking to the tumor core and, as a result, inhibit tumor killing. The production of reactive nitrogen species (RNS) within the tumor microenvironment has been reported in mouse and human cancers. We describe a novel RNS-dependent posttranslational modification of chemokines that has a profound impact on leukocyte recruitment to mouse and human tumors. Intratumoral RNS production induces CCL2 chemokine nitration and hinders T cell infiltration, resulting in the trapping of tumor-specific T cells in the stroma that surrounds cancer cells. Preconditioning of the tumor microenvironment with novel drugs that inhibit CCL2 modification facilitates CTL invasion of the tumor, suggesting that these drugs may be effective in cancer immunotherapy. Our results unveil an unexpected mechanism of tumor evasion and introduce new avenues for cancer immunotherapy.

CCL2 and interleukin (IL)-6 are among the most prevalent cytokines in the tumor microenvironment, with expression generally correlating with tumor progression and metastasis. CCL2 and IL-6 induced expression of each other in CD11b(+) cells isolated from human peripheral blood. It was demonstrated that both cytokines induce up-regulation of the antiapoptotic proteins cFLIP(L) (cellular caspase-8 (FLICE)-like inhibitory protein), Bcl-2, and Bcl-X(L) and inhibit the cleavage of caspase-8 and subsequent activation of the caspase-cascade, thus protecting cells from apoptosis under serum deprivation stress. Furthermore, both cytokines induced hyperactivation of autophagy in these cells. Upon CCL2 or IL-6 stimulation, CD11b(+) cells demonstrated a significant increase in the mannose receptor (CD206) and the CD14(+)/CD206(+) double-positive cells, suggesting a polarization of macrophages toward the CD206(+) M2-type phenotype. Caspase-8 inhibitors mimicked the cytokine-induced up-regulation of autophagy and M2 polarization. Furthermore, E64D and leupeptin, which are able to function as inhibitors of autophagic degradation, reversed the effect of caspase-8 inhibitors in the M2-macrophage polarization, indicating a role of autophagy in this mechanism. Additionally, in patients with advanced castrate-resistant prostate cancer, metastatic lesions exhibited an increased CD14(+)/CD206(+) double-positive cell population compared with normal tissues. Altogether, these findings suggest a role for CCL2 and IL-6 in the survival of myeloid monocytes recruited to the tumor microenvironment and their differentiation toward tumor-promoting M2-type macrophages via inhibition of caspase-8 cleavage and enhanced autophagy.

Our previous study showed that activation of c-jun-N-terminal kinase (JNK) in spinal astrocytes plays an important role in neuropathic pain sensitization. We further investigated how JNK regulates neuropathic pain. In cultured astrocytes, tumor necrosis factor alpha (TNF-alpha) transiently activated JNK via TNF receptor-1. Cytokine array indicated that the chemokine CCL2/MCP-1 (monocyte chemoattractant protein-1) was strongly induced by the TNF-alpha/JNK pathway. MCP-1 upregulation by TNF-alpha was dose dependently inhibited by the JNK inhibitors SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one) and D-JNKI-1. Spinal injection of TNF-alpha produced JNK-dependent pain hypersensitivity and MCP-1 upregulation in the spinal cord. Furthermore, spinal nerve ligation (SNL) induced persistent neuropathic pain and MCP-1 upregulation in the spinal cord, and both were suppressed by D-JNKI-1. Remarkably, MCP-1 was primarily induced in spinal cord astrocytes after SNL. Spinal administration of MCP-1 neutralizing antibody attenuated neuropathic pain. Conversely, spinal application of MCP-1 induced heat hyperalgesia and phosphorylation of extracellular signal-regulated kinase in superficial spinal cord dorsal horn neurons, indicative of central sensitization (hyperactivity of dorsal horn neurons). Patch-clamp recordings in lamina II neurons of isolated spinal cord slices showed that MCP-1 not only enhanced spontaneous EPSCs but also potentiated NMDA- and AMPA-induced currents. Finally, the MCP-1 receptor CCR2 was expressed in neurons and some non-neuronal cells in the spinal cord. Together, we have revealed a previously unknown mechanism of MCP-1 induction and action. MCP-1 induction in astrocytes after JNK activation contributes to central sensitization and neuropathic pain facilitation by enhancing excitatory synaptic transmission. Inhibition of the JNK/MCP-1 pathway may provide a new therapy for neuropathic pain management.

A causal role was recently attributed to inflammation in many malignant diseases, including breast cancer. The different inflammatory mediators that are involved in this disease include cells, cytokines and chemokines. Of these, many studies have addressed the involvement and roles of the inflammatory chemokines CCL2 (MCP-1) and CCL5 (RANTES) in breast malignancy. While minimally expressed by normal breast epithelial duct cells, both chemokines are highly expressed by breast tumor cells at primary tumor sites, indicating that CCL2 and CCL5 expression is acquired in the course of malignant transformation, and suggesting that the two chemokines play a role in breast cancer development and/or progression. Supporting this possibility are findings showing significant associations between CCL2 and CCL5 and more advanced disease course and progression. Furthermore, studies in animal model systems have shown active and causative roles for the two chemokines in this disease. In line with the tumor-promoting roles of CCL2 and CCL5 in breast cancer, the two chemokines were shown to mediate many types of tumor-promoting cross-talks between the tumor cells and cells of the tumor microenvironment: (1) they shift the balance at the tumor site between different leukocyte cell types by increasing the presence of deleterious tumor-associated macrophages (TAM) and inhibiting potential anti-tumor T cell activities; (2) of the two chemokines, mainly CCL2 promotes angiogenesis; (3) CCL2 and CCL5 which are expressed by cells of the tumor microenvironment osteoblasts and mesenchymal stem cells play a role in breast metastatic processes. In addition, both chemokines act directly on the tumor cells to promote their pro-malignancy phenotype, by increasing their migratory and invasion-related properties. Together, the overall current information suggests that CCL2 and CCL5 are inflammatory mediators with pro-malignancy activities in breast cancer, and that they should be considered as potential therapeutic targets for the limitation of this disease.

Monocyte-derived macrophages are essential for recovery after spinal cord injury, but their homing mechanism is poorly understood. Here, we show that although of common origin, the homing of proinflammatory (M1) and the "alternatively activated" anti-inflammatory (M2) macrophages to traumatized spinal cord (SC) was distinctly regulated, neither being through breached blood-brain barrier. The M1 macrophages (Ly6c(hi)CX3CR1(lo)) derived from monocytes homed in a CCL2 chemokine-dependent manner through the adjacent SC leptomeninges. The resolving M2 macrophages (Ly6c(lo)CX3CR1(hi)) derived from monocytes trafficked through a remote blood-cerebrospinal-fluid (CSF) barrier, the brain-ventricular choroid plexus (CP), via VCAM-1-VLA-4 adhesion molecules and epithelial CD73 enzyme for extravasation and epithelial transmigration. Blockage of these determinants, or mechanical CSF flow obstruction, inhibited M2 macrophage recruitment and impaired motor-function recovery. The CP, along with the CSF and the central canal, provided an anti-inflammatory supporting milieu, potentially priming the trafficking monocytes. Overall, our finding demonstrates that the route of monocyte entry to central nervous system provides an instructional environment to shape their function.

Millions of people worldwide suffer from neuropathic pain as a result of damage to or dysfunction of the nervous system under various disease conditions. Development of effective therapeutic strategies requires a better understanding of molecular and cellular mechanisms underlying the pathogenesis of neuropathic pain. It has been increasingly recognized that spinal cord glial cells such as microglia and astrocytes play a critical role in the induction and maintenance of neuropathic pain by releasing powerful neuromodulators such as proinflammatory cytokines and chemokines. Recent evidence reveals chemokines as new players in pain control. In this article, we review evidence for chemokine modulation of pain via neuronal-glial interactions by focusing on the central role of two chemokines, CX3CL1 (fractalkine) and CCL2 (MCP-1), because they differentially regulate neuronal-glial interactions. Release of CX3CL1 from neurons is ideal to mediate neuronal-to-microglial signaling, since the sole receptor of this chemokine, CX3CR1, is expressed in spinal microglia and activation of the receptor leads to phosphorylation of p38 MAP kinase in microglia. Although CCL2 was implicated in neuronal-to-microglial signaling, a recent study shows a novel role of CCL2 in astroglial-to-neuronal signaling after nerve injury. In particular, CCL2 rapidly induces central sensitization by increasing the activity of NMDA receptors in dorsal horn neurons. Insights into the role of chemokines in neuronal-glial interactions after nerve injury will identify new targets for therapeutic intervention of neuropathic pain.

Cytokines and microglia have been implicated in anxiety, depression, neurodegeneration as well as the regulation of alcohol drinking and other consumatory behaviors, all of which are associated with alcoholism. Studies using animal models of alcoholism suggest that microglia and proinflammatory cytokines contribute to alcoholic pathologies [Crews, F.T., Bechara, R., Brown, L.A., Guidot, D.M., Mandrekar, P., Oak, S., Qin, L., Szabo, G., Wheeler, M., Zou, J., (2006) Cytokines and alcohol. Alcohol., Clin. Exp. Res. 30:720-730]. In the current study, human postmortem brains from moderate drinking controls and alcoholics obtained from the New South Wales Tissue Resource Center were used to study the cytokine, monocyte chemoattractant protein 1 (MCP-1,CCL2) and microglia markers in various brain regions. Since MCP-1 is a key proinflammatory cytokine induced by chronic alcohol treatment of mice, and known to regulate drinking behavior in mice, MCP-1 protein levels from human brain homogenate were measured using ELISA, and indicated increased MCP-1 concentration in ventral tegmental area (VTA), substantia nigra (SN), hippocampus and amygdala of alcoholic brains as compared with controls. Immunohistochemistry was further performed to visualize human microglia using ionized calcium binding adaptor protein-1 (Iba-1), and Glucose transporter-5 (GluT5). Alcoholics were found to have brain region-specific increases in microglial markers. In cingulate cortex, both Iba-1 and GluT5 were increased in alcoholic brains relative to controls. Alternatively, no detectable change was found in amygdala nuclei. In VTA and midbrain, only GluT5, but not Iba-1 was increased in alcoholic brains. These data suggest that the enhanced expression of MCP-1 and microglia activities in alcoholic brains could contribute to ethanol-induced pathogenesis.

CD16+ monocytes represent 5-10% of peripheral blood monocytes in normal individuals and are dramatically expanded in several pathological conditions including sepsis, human immunodeficiency virus 1 infection, and cancer. CD16+ monocytes produce high levels of proinflammatory cytokines and may represent dendritic cell precursors in vivo. The mechanisms that mediate the recruitment of CD16+ monocytes into tissues remain unknown. Here we investigate molecular mechanisms of CD16+ monocyte trafficking and show that migration of CD16+ and CD16- monocytes is mediated by distinct combinations of adhesion molecules and chemokine receptors. In contrast to CD16- monocytes, CD16+ monocytes expressed high CX3CR1 and CXCR4 but low CCR2 and CD62L levels and underwent efficient transendothelial migration in response to fractalkine (FKN; FKN/CX3CL1) and stromal-derived factor 1 alpha (CXCL12) but not monocyte chemoattractant protein 1 (CCL2). CD16+ monocytes arrested on cell surface-expressed FKN under flow with higher frequency compared with CD16- monocytes. These results demonstrate that FKN preferentially mediates arrest and migration of CD16+ monocytes and suggest that recruitment of this proinflammatory monocyte subset to vessel walls via the CX3CR1-FKN pathway may contribute to vascular and tissue injury during pathological conditions.

Chemokines play well established roles as attractants of naïve and effector T cells. New studies indicate that chemokines also have roles in regulating T cell differentiation. Blocking Gi protein-coupled receptor signaling by pertussis toxin as well as deficiencies in G alpha 12, chemokine receptor 2 (CCR2), CCR5, chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein 1, or MCP-1), CCL3 (macrophage inflammatory protein 1 alpha, or MIP-1 alpha) and CCL5 (RANTES) have all been found to have effects on the magnitude and cytokine polarity of the T cell response. Here we focus on findings in the CCL2-CCR2 and CCL3-CCR5 ligand-receptor systems. The roles of these molecules in regulating T cell fate include possible indirect effects on antigen-presenting cells and direct effects on differentiating T cells. Models to account for the action of chemokines and G protein-coupled receptor signals in regulating T cell differentiation are discussed.

Microglia, the primary immune effector cells in the brain, continually monitor the tissue parenchyma for pathological alterations and become activated in Alzheimer's disease. Loss of signaling between neurons and microglia via deletion of the microglial receptor, CX3CR1, worsens phenotypes in various models of neurodegenerative diseases. In contrast, CX3CR1 deficiency ameliorates pathology in murine stroke models. To examine the role of CX3CR1 in Alzheimer's disease-related β-amyloid pathology, we generated APPPS1 and R1.40 transgenic mouse models of Alzheimer's disease deficient for CX3CR1. Surprisingly, CX3CR1 deficiency resulted in a gene dose-dependent reduction in β-amyloid deposition in both the APPPS1 and R1.40 mouse models of AD. Immunohistochemical analysis revealed reduced staining for CD68, a marker of microglial activation. Furthermore, quantitative immunohistochemical analysis revealed reduced numbers of microglia surrounding β-amyloid deposits in the CX3CR1-deficient APPPS1 animals. The reduced β-amyloid pathology correlated with reduced levels of TNFα and CCL2 mRNAs, but elevated IL1β mRNA levels, suggesting an altered neuroinflammatory milieu. Finally, to account for these seemingly disparate results, both in vitro and in vivo studies provided evidence that CX3CL1/CX3CR1 signaling alters the phagocytic capacity of microglia, including the uptake of Aβ fibrils. Taken together, these results demonstrate that loss of neuron-microglial fractalkine signaling leads to reduced β-amyloid deposition in mouse models of AD that is potentially mediated by altered activation and phagocytic capability of CX3CR1-deficient microglia.

BACKGROUND

Understanding the earliest central nervous system (CNS) events during human immunodeficiency virus (HIV) infection is crucial to knowledge of neuropathogenesis, but these have not previously been described in humans.

METHODS

Twenty individuals who had acute HIV infection (Fiebig stages I-IV), with average 15 days after exposure, underwent clinical neurological, cerebrospinal fluid (CSF), magnetic resonance imaging, and magnetic resonance spectroscopy (MRS) characterization.

RESULTS

HIV RNA was detected in the CSF from 15 of 18 subjects as early as 8 days after estimated HIV transmission. Undetectable CSF levels of HIV (in 3 of 18) was noted during Fiebig stages I, II, and III, with plasma HIV RNA levels of 285651, 2321, and 81978 copies/mL, respectively. On average, the CSF HIV RNA level was 2.42 log(10) copies/mL lower than that in plasma. There were no cases in which the CSF HIV RNA level exceeded that in plasma. Headache was common during the acute retroviral syndrome (in 11 of 20 subjects), but no other neurological signs or symptoms were seen. Intrathecal immune activation was identified in some subjects with elevated CSF neopterin, monocyte chemotactic protein/CCL2, and interferon γ-induced protein 10/CXCL-10 levels. Brain inflammation was suggested by MRS.

CONCLUSIONS

CSF HIV RNA was detectable in humans as early as 8 days after exposure. CNS inflammation was apparent by CSF analysis and MRS in some individuals during acute HIV infection.

Sustained hepatic inflammation is an important factor in progression of chronic liver diseases, including hepatitis C or non-alcoholic steatohepatitis. Liver inflammation is regulated by chemokines, which regulate the migration and activities of hepatocytes, Kupffer cells, hepatic stellate cells, endothelial cells, and circulating immune cells. However, the effects of the different chemokines and their receptors vary during pathogenesis of different liver diseases. During development of chronic viral hepatitis, CCL5 and CXCL10 regulate the cytopathic versus antiviral immune responses of T cells and natural killer cells. During development of nonalcoholic steatohepatitis, CCL2 and its receptor are up-regulated in the liver, where they promote macrophage accumulation, inflammation, fibrosis, and steatosis, as well as in adipose tissue. CCL2 signaling thereby links hepatic and systemic inflammation related to metabolic disorders and insulin resistance. Several chemokine signaling pathways also promote hepatic fibrosis. Recent studies have shown that other chemokines and immune cells have anti-inflammatory and antifibrotic activities. Chemokines and their receptors can also contribute to the pathogenesis of hepatocellular carcinoma, promoting proliferation of cancer cells, the inflammatory microenvironment of the tumor, evasion of the immune response, and angiogenesis. We review the roles of different chemokines in the pathogenesis of liver diseases and their potential use as biomarkers or therapeutic targets.

Secretion of C-C chemokine ligand 2 (CCL2) by mammary tumours recruits CCR2-expressing inflammatory monocytes to primary tumours and metastatic sites, and CCL2 neutralization in mice inhibits metastasis by retaining monocytes in the bone marrow. Here we report a paradoxical effect of CCL2 in four syngeneic mouse models of metastatic breast cancer. Surprisingly, interruption of CCL2 inhibition leads to an overshoot of metastases and accelerates death. This is the result of monocyte release from the bone marrow and enhancement of cancer cell mobilization from the primary tumour, as well as blood vessel formation and increased proliferation of metastatic cells in the lungs in an interleukin (IL)-6- and vascular endothelial growth factor (VEGF)-A-dependent manner. Notably, inhibition of CCL2 and IL-6 markedly reduced metastases and increased survival of the animals. CCL2 has been implicated in various neoplasias and adopted as a therapeutic target. However, our results call for caution when considering anti-CCL2 agents as monotherapy in metastatic disease and highlight the tumour microenvironment as a critical determinant of successful anti-metastatic therapy.

Amyotrophic lateral sclerosis (ALS) is a progressive disease associated with neuronal cell death that is thought to involve aberrant immune responses. Here we investigated the role of innate immunity in a mouse model of ALS. We found that inflammatory monocytes were activated and that their progressive recruitment to the spinal cord, but not brain, correlated with neuronal loss. We also found a decrease in resident microglia in the spinal cord with disease progression. Prior to disease onset, splenic Ly6Chi monocytes expressed a polarized macrophage phenotype (M1 signature), which included increased levels of chemokine receptor CCR2. As disease onset neared, microglia expressed increased CCL2 and other chemotaxis-associated molecules, which led to the recruitment of monocytes to the CNS by spinal cord-derived microglia. Treatment with anti-Ly6C mAb modulated the Ly6Chi monocyte cytokine profile, reduced monocyte recruitment to the spinal cord, diminished neuronal loss, and extended survival. In humans with ALS, the analogous monocytes (CD14+CD16-) exhibited an ALS-specific microRNA inflammatory signature similar to that observed in the ALS mouse model, linking the animal model and the human disease. Thus, the profile of monocytes in ALS patients may serve as a biomarker for disease stage or progression. Our results suggest that recruitment of inflammatory monocytes plays an important role in disease progression and that modulation of these cells is a potential therapeutic approach.

Innate responses in the CNS are critical to first line defense against infection and injury. Leukocytes migrate to inflammatory sites in response to chemokines. We studied leukocyte migration and glial chemokine expression within the denervated hippocampus in response to axonal injury caused by entorhinodentate lesions. A population of Mac1/CD11b+ CD45high macrophages (distinct from CD45low microglia) was specifically detected within the lesion-reactive hippocampus by 12 hr after injury. Significant infiltration by CD3+ T cells did not occur in the denervated hippocampus until 24 hr after axotomy. A broad spectrum of chemokines [RANTES/CCL5, monocyte chemoattractant protein (MCP)-1/CCL2, interferon gamma inducible protein (IP)-10/CXCL10, macrophage inflammatory protein (MIP)-1alpha/CCL3, MIP-1beta/CCL4, and MIP-2/CXCL2] was induced at this time. RANTES/CCL5 was not significantly elevated until 24 hr after axotomy, whereas MCP-1/CCL2 was significantly induced before leukocyte infiltration occurred. Neither T cells nor macrophages infiltrated the denervated hippocampus of CCR2-deficient mice, arguing for a critical role for the CCR2 ligand MCP-1/CCL2 in leukocyte migration. Both T cells and macrophages infiltrated CCR5-deficient hippocampi, showing that CCR5 ligands (including RANTES/CCL5) are not critical to this response. In situ hybridization combined with immunohistochemistry for ionized binding calcium adapter molecule (iba)1 or glial fibrillary acidic protein (GFAP) identified iba1+ microglia and GFAP+ astrocytes as major sources of MCP-1/CCL2 within the lesion-reactive hippocampus. We conclude that leukocyte responses to CNS axonal injury are directed via innate glial production of chemokines.

In a lethal West Nile virus (WNV) model, central nervous system infection triggered a threefold increase in CD45(int)/CD11b(+)/CD11c(-) microglia at days 6-7 postinfection (p.i.). Few microglia were proliferating, suggesting that the increased numbers were derived from a migratory precursor cell. Depletion of "circulating" (Gr1(-)(Ly6C(lo))CX3CR1(+)) and "inflammatory" (Gr1(hi)/Ly6C(hi)/CCR2(+)) classical monocytes during infection abrogated the increase in microglia. C57BL/6 chimeras reconstituted with cFMS-enhanced green fluorescent protein (EGFP) bone marrow (BM) showed large numbers of peripherally derived (GFP(+)) microglia expressing GR1(+)(Ly6C(+)) at day 7 p.i., suggesting that the inflammatory monocyte is a microglial precursor. This was confirmed by adoptive transfer of labeled BM (Ly6C(hi)/CD115(+)) or circulating inflammatory monocytes that trafficked to the WNV-infected brain and expressed a microglial phenotype. CCL2 is a chemokine that is highly expressed during WNV infection and important in inflammatory monocyte trafficking. Neutralization of CCL2 not only reduced the number of GFP(+) microglia in the brain during WNV infection but prolonged the life of infected animals. Therefore, CCL2-dependent inflammatory monocyte migration is critical for increases in microglia during WNV infection and may also play a pathogenic role during WNV encephalitis.

Biofilms are complex communities of bacteria encased in a matrix composed primarily of polysaccharides, extracellular DNA, and protein. Staphylococcus aureus can form biofilm infections, which are often debilitating due to their chronicity and recalcitrance to antibiotic therapy. Currently, the immune mechanisms elicited during biofilm growth and their impact on bacterial clearance remain to be defined. We used a mouse model of catheter-associated biofilm infection to assess the functional importance of TLR2 and TLR9 in the host immune response during biofilm formation, because ligands for both receptors are present within the biofilm. Interestingly, neither TLR2 nor TLR9 impacted bacterial density or inflammatory mediator secretion during biofilm growth in vivo, suggesting that S. aureus biofilms circumvent these traditional bacterial recognition pathways. Several potential mechanisms were identified to account for biofilm evasion of innate immunity, including significant reductions in IL-1β, TNF-α, CXCL2, and CCL2 expression during biofilm infection compared with the wound healing response elicited by sterile catheters, limited macrophage invasion into biofilms in vivo, and a skewing of the immune response away from a microbicidal phenotype as evidenced by decreases in inducible NO synthase expression concomitant with robust arginase-1 induction. Coculture studies of macrophages with S. aureus biofilms in vitro revealed that macrophages successful at biofilm invasion displayed limited phagocytosis and gene expression patterns reminiscent of alternatively activated M2 macrophages. Collectively, these findings demonstrate that S. aureus biofilms are capable of attenuating traditional host proinflammatory responses, which may explain why biofilm infections persist in an immunocompetent host.

Multiple sclerosis (MS) is the commonest inflammatory demyelinating disease of the human central nervous system (CNS). In MS, CNS inflammation is associated with demyelination and axonal degeneration, which leads to clinical presentation. Expression and cellular localization of CCL2/MCP-1 and CCR2 in MS have been described in the three compartments: brain, cerebrospinal fluid (CSF) and blood. Evidence from descriptive, transgenic, knockout and neutralizing studies of experimental autoimmune encephalomyelitis (EAE) points towards a nonredundant role of CCL2 and CCR2 in the recruitment of inflammatory infiltrate into the CNS. Hence, CCL2 and CCR2 may be targets for specific and effective treatment in MS.

OBJECTIVE

To elucidate the role of systemic inflammation in nonalcoholic fatty liver disease (NAFLD).

METHODS

Serum samples in 47 patients with histologically verified NAFLD (22 with simple steatosis and 25 with nonalcoholic steatohepatitis [NASH]), and in 30 age-, sex- and ethnicity-matched healthy controls, were assessed for (i) general markers of inflammation (C-reactive protein [CRP], tumor necrosis factor [TNF]-alpha, and interleukin [IL]-6), (ii) chemokines (CC-chemokine ligand [CCL] 2/monocyte chemoattractant protein [MCP]-1, CCL19 and <em>CCL2</em>1), (iii) adipocytokines related to insulin resistance and inflammation (adiponectin and leptin) and (iv) a marker of oxidative stress (8-isoprostane-F2alpha).

RESULTS

Serum levels of several inflammatory cytokines were increased in NAFLD as compared to controls, and IL-6 (P=0.017), CCL2/MCP-1 (P=0.008) and CCL19 (P=0.001), but not CRP (P=0.199), remained elevated also after correction for sex, body mass index (BMI) and age. Comparing NASH with simple steatosis, levels of TNF-alpha (P=0.024) and CCL2/MCP-1 (P=0.012) were elevated and adiponectin (in women) (P=0.001) were decreased also after adjustment for sex, BMI and presence of the metabolic syndrome.

CONCLUSIONS

Our results indicate that patients with NAFLD are characterized by a low-grade systemic inflammation. The high CCL2/MCP-1 levels in NASH might be of importance for the conversion from simple steatosis to NASH.