Immune responses in the central nervous system (CNS) are carefully regulated. Despite the absence of most immune processes and a substantive blood brain barrier, potent immune responses form during infection and autoimmunity. Astrocytes are innate immune sentinels that ensheath parenchymal blood vessels and sit at the gateway to the CNS parenchyma. Viral and bacterial infections trigger the influx of distinct leukocyte subsets. We show that astrocytes alone are sufficient for distinguishing between these two main types of infection and triggers release of relevant chemokines that relate to the microbe recognised. Bacterial-associated molecules induced the preferential expression of CCL2, CXCL1, CCL20 and CCL3 whilst a virus-associated dsRNA analogue preferentially up-regulated CXCL10 and CCL5. Thus, astrocytes can respond to infection in a distinct and appropriate manner suggesting they have the capacity to attract appropriate sets of leukocytes into the brain parenchyma. Astrocytes themselves are unable to respond to these chemokines since they were devoid of most chemokine receptors but expressed CXCR4, CXCR7 and CXCR6 at rest. Stimulation with TGF-beta specifically up-regulated CXCR6 expression and may explain how TGF-beta/CXCL16-expressing gliomas are so effective at attracting astroglial cells.

Exacerbated inflammation in renal ischemia-reperfusion injury, the major cause of intrinsic acute renal failure, is a key trigger of kidney damage. During disease endogenous danger signals stimulate innate immune cells via Toll-like receptors (TLR)-2 and -4 and accelerate inflammatory responses. Here we show that production of soluble biglycan, a small leucine-rich proteoglycan, is induced during reperfusion and that it functions as endogenous agonist of TLR-2/4. Biglycan-mediated activation of TLR-2/4 initiates an inflammatory response in native kidneys, which is marked by the release of cytokines and chemokines and recruitment of inflammatory cells. Overexpression of soluble circulating biglycan before ischemic reperfusion enhanced plasma and renal levels of TNF-α, CXCL1, CCL2 and CCL5, caused influx of neutrophils, macrophages and T cells and overall worsened renal function in wild type mice. We provide robust genetic evidence for TLR-2/4 requirement insofar as biglycan biological effects were markedly dampened in mice deficient in both innate immune receptors, Tlr2(-/-);Tlr4(-/-) mice. Thus, signaling of soluble biglycan via TLR-2/4 could represent a novel therapeutic target for the prevention and possible treatment of patients with acute renal ischemia-reperfusion injury.

The cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway detects cytosolic DNA to activate innate immune responses. Poly(ADP-ribose) polymerase inhibitors (PARPi) selectively target cancer cells with DNA repair deficiencies such as those caused by BRCA1 mutations or ERCC1 defects. Using isogenic cell lines and patient-derived samples, we showed that ERCC1-defective non-small cell lung cancer (NSCLC) cells exhibit an enhanced type I IFN transcriptomic signature and that low ERCC1 expression correlates with increased lymphocytic infiltration. We demonstrated that clinical PARPi, including olaparib and rucaparib, have cell-autonomous immunomodulatory properties in ERCC1-defective NSCLC and BRCA1-defective triple-negative breast cancer (TNBC) cells. Mechanistically, PARPi generated cytoplasmic chromatin fragments with characteristics of micronuclei; these were found to activate cGAS/STING, downstream type I IFN signaling, and CCL5 secretion. Importantly, these effects were suppressed in PARP1-null TNBC cells, suggesting that this phenotype resulted from an on-target effect of PARPi on PARP1. PARPi also potentiated IFN-γ-induced PD-L1 expression in NSCLC cell lines and in fresh patient tumor cells; this effect was enhanced in ERCC1-deficient contexts. Our data provide a preclinical rationale for using PARPi as immunomodulatory agents in appropriately molecularly selected populations.

Immune responses against cancer rely upon leukocyte trafficking patterns that are coordinated by chemokines. CCR5, the receptor for chemotactic chemokines MIP1alpha, MIP1beta, and RANTES (CCL3, CCL4, CCL5), exerts major regulatory effects on CD4(+)- and CD8(+) T cell-mediated immunity. Although CCR5 and its ligands participate in the response to various pathogens, its relevance to tumoral immune control has been debated. Here, we report that CCR5 has a specific, ligand-dependent role in optimizing antitumor responses. In adoptive transfer studies, efficient tumor rejection required CCR5 expression by both CD4(+) and CD8(+) T cells. CCR5 activation in CD4(+) cells resulted in CD40L upregulation, leading to full maturation of antigen-presenting cells and enhanced CD8(+) T-cell crosspriming and tumor infiltration. CCR5 reduced chemical-induced fibrosarcoma incidence and growth, but did not affect the onset or progression of spontaneous breast cancers in tolerogenic Tg(MMTV-neu) mice. However, CCR5 was required for TLR9-mediated reactivation of antineu responses in these mice. Our results indicate that CCR5 boosts T-cell responses to tumors by modulating helper-dependent CD8(+) T-cell activation.

OBJECTIVE

High expression of galectin 3 at sites of joint destruction in rheumatoid arthritis (RA) suggests that galectin 3 plays a role in RA pathogenesis. Previous studies have demonstrated the effects of galectins on immune cells, such as lymphocytes and macrophages. This study was undertaken to investigate the hypothesis that galectin 3 induces proinflammatory effects in RA by modulating the pattern of cytokine and chemokine production in synovial fibroblasts.

METHODS

Matched samples of RA synovial and skin fibroblasts were pretreated with galectin 3 or tumor necrosis factor alpha (TNFalpha), and the levels of a panel of cytokines, chemokines, and matrix metalloproteinases (MMPs) were determined using enzyme-linked immunosorbent assays and multiplex assays. Specific inhibitors were used to dissect signaling pathways, which were confirmed by Western blotting and NF-kappaB activation assay.

RESULTS

Galectin 3 induced secretion of interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor, CXCL8, and MMP-3 in both synovial and skin fibroblasts. By contrast, galectin 3-induced secretion of TNFalpha, CCL2, CCL3, and CCL5 was significantly greater in synovial fibroblasts than in skin fibroblasts. TNFalpha blockade ruled out autocrine TNFalpha-stimulated induction of chemokines. The MAPKs p38, JNK, and ERK were necessary for IL-6 production, but phosphatidylinositol 3-kinase (PI 3-kinase) was required for selective CCL5 induction. NF-kappaB activation was required for production of both IL-6 and CCL5.

CONCLUSIONS

Our findings indicate that galectin 3 promotes proinflammatory cytokine secretion by tissue fibroblasts. However, galectin 3 induces the production of mononuclear cell-recruiting chemokines uniquely from synovial fibroblasts, but not matched skin fibroblasts, via a PI 3-kinase signaling pathway. These data provide further evidence of the role of synovial fibroblasts in regulating the pattern and persistence of the inflammatory infiltrate in RA and suggest a new and important functional consequence of the observed high expression of galectin 3 in the rheumatoid synovium.

West Nile (WN) virus is a mosquito-borne flavivirus that can cause lethal encephalitis in humans and horses. The WN virus endemic in New York City (NY) in 1999 caused large-scale mortality of wild birds that was not evident in endemic areas in other parts of the world, and the pathogenesis of the WN virus strain isolated in NY (NY strain) appears to differ from that of previously isolated strains. However, the pathogenesis of NY strain infection remains unclear. This study examined CC (RANTES/CCL5, MIP-1 alpha/CCL3, MIP-1 beta/CCL4) and CXC (IP-10/CXCL10, B lymphocyte chemoattractant (BLC/CXCL13), and B cell- and monocyte-activating chemokine (BMAC/CXCL14)) chemokine expression during lethal NY strain and non-lethal Eg101 strain infection in mice. We found that the mRNA of the CC chemokines, RANTES, MIP-1 alpha, MIP-1 beta, and IP-10 was highly up-regulated in the brain of NY strain-infected mice. By contrast, BLC mRNA was not detected in either group of mice, and BMAC mRNA was highly up-regulated in late stage of infection with the non-lethal Eg101 strain relative to levels in NY strain-infected mice.

OBJECTIVE

Chemokines initiate the immune response by controlling leukocyte migration and lymphocyte development. Macrophage infiltration of the decidua has been implicated in the genesis of recurrent miscarriage and preeclampsia. Therefore, we determined whether cultured human decidual cells produce monocyte/macrophage-recruiting chemokines in response to a potent pro-inflammatory cytokine, interleukin-1beta (IL-1beta), and whether decidual cell-conditioned medium contains monocyte- and macrophage-chemoattractant activity.

METHODS

Leukocyte-free first trimester decidual cells were treated for 6h with estradiol (E(2)) and medroxyprogesterone acetate (MPA) to mimic the steroidal milieu of pregnancy, or E(2) and MPA and IL-1beta (1 ng/ml) to mimic inflamed decidua. Total RNA was used for cDNA synthesis. Biotinylated cRNAs were generated and chemically fragmented for hybridization on Affymetrix HG_U133 Plus 2.0 chips followed by fluorescence labeling and optical scanning. Raw data generated from Affymetrix GCOS 1.2 (GeneChip Operating Software) were analyzed by GeneSpring 7.2 software. Subsequently microarray results were validated by real time RT-PCR and Western blotting. A functional study of monocyte migration was carried out also using conditioned media from culture.

RESULTS

Five chemokines responsible for monocyte/macrophage chemoattraction and activation, including C-C motif ligand 2 (CCL2), CCL5, C-X-C motif ligand 2 (CXCL2), CXCL3 and CXCL8, were markedly elevated from 29- to 975-fold after exposure to IL-1beta in cultured first trimester decidual cells. The results of real-time RT-PCR (up-regulation from 43- to 3069-fold) and Western blotting (up-regulation from 15- to 300-fold) confirmed the microarray findings. Monocyte migration was significantly induced by the conditioned medium from IL-1beta-treated decidual cells.

CONCLUSIONS

Treatment of first trimester decidual cells with IL-1beta induces secretion of monocyte/macrophage recruiting-chemokines and promotes monocyte migration. Extrapolation of these in vitro results to the milieu of implantation site suggests a mechanism whereby IL-1beta could mediate excessive macrophage infiltration of the decidua.

Lung epithelial cells are the primary cellular targets for respiratory virus pathogens such as influenza and parainfluenza viruses. Here, we have analyzed influenza A, influenza B and Sendai virus-induced chemokine response in human A549 lung epithelial cells. Influenza virus infection resulted in low CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8 and CXCL10/IP-10 production at late times of infection. However, when cells were pretreated with TNF-alpha or IFN-alpha, influenza-A-virus-induced chemokine production was greatly enhanced. Cytokine pretreatment resulted in enhanced expression of RIG-I, IKKepsilon, interferon regulatory factor (IRF)1, IRF7 and p50 proteins. Most importantly, influenza-A-virus-induced DNA binding of IRF1, IRF3, IRF7 and NF-kappaB onto CXCL10 ISRE and NF-kappaB elements, respectively, was markedly enhanced in cytokine-pretreated cells. Our results suggest that IFN-alpha and TNF-alpha have a significant role in priming epithelial cells for higher cytokine and chemokine production in influenza A virus infection.

BACKGROUND

Breast cancer progression is promoted by stromal cells that populate the tumors, including cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs). The activities of CAFs and MSCs in breast cancer are integrated within an intimate inflammatory tumor microenvironment (TME) that includes high levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Here, we identified the impact of TNF-α and IL-1β on the inflammatory phenotype of CAFs and MSCs by determining the expression of inflammatory chemokines that are well-characterized as pro-tumorigenic in breast cancer: CCL2 (MCP-1), CXCL8 (IL-8) and CCL5 (RANTES).

METHODS

Chemokine expression was determined in breast cancer patient-derived CAFs by ELISA and in patient biopsies by immunohistochemistry. Chemokine levels were determined by ELISA in (1) human bone marrow-derived MSCs stimulated by tumor conditioned media (Tumor CM) of breast tumor cells (MDA-MB-231 and MCF-7) at the end of MSC-to-CAF-conversion process; (2) Tumor CM-derived CAFs, patient CAFs and MSCs stimulated by TNF-α (and IL-1β). The roles of AP-1 and NF-κB in chemokine secretion were analyzed by Western blotting and by siRNAs to c-Jun and p65, respectively. Migration of monocytic cells was determined in modified Boyden chambers.

RESULTS

TNF-α (and IL-1β) induced the release of CCL2, CXCL8 and CCL5 by MSCs and CAFs generated by prolonged stimulation of MSCs with Tumor CM of MDA-MB-231 and MCF-7 cells. Patient-derived CAFs expressed CCL2 and CXCL8, and secreted CCL5 following TNF-α (and IL-1β) stimulation. CCL2 was expressed in CAFs residing in proximity to breast tumor cells in biopsies of patients diagnosed with invasive ductal carcinoma. CCL2 release by TNF-α-stimulated MSCs was mediated by TNF-RI and TNF-RII, through the NF-κB but not via the AP-1 pathway. Exposure of MSCs to TNF-α led to potent CCL2-induced migration of monocytic cells, a process that may yield pro-cancerous myeloid infiltrates in breast tumors.

CONCLUSIONS

Our novel results emphasize the important roles of inflammation-stroma interactions in breast cancer, and suggest that NF-κB may be a potential target for inhibition in tumor-adjacent stromal cells, enabling improved tumor control in inflammation-driven malignancies.

The expression of chemokine receptors and chemokine production by adult human non-transformed astrocytes, grade III astrocytoma and grade IV glioblastoma tumour cell lines were determined. Here, we show an increased expression of CXCR3 and CXCR4, and a decreased expression of CXCR1 and CCR4 by glioma cells compared to adult human astrocytes. Glioma cells showed increased production of CXCL10, whereas production of other chemokines was decreased (CXCL8, CCL2, CCL5, and CCL22). CXCL10 induced an ERK1/2-dependent increase in [(3)H] thymidine uptake. These results suggest that expression of chemokine receptor/ligand pairs such as CXCR3/CXCL10 have an important role in the proliferation of glioma cells.

Imbalance oxidative stress and chemokines are considered as a universal factors involved in the development of various clinical features seen in the patients with SLE and arthritis. To evaluate the interaction between oxidative stress and chemokines and their relationship with disease activity in SLE and RA patients, oxidative/anti-oxidant profiles and chemokines were assessed. Oxidant and anti-oxidant enzymes were measured in the plasma and the levels of chemokines; MCP-1/CCL2, RANTES/CCL5, MIP-1β/CCL-4 and IP-10/CXCL-10 were evaluated in the serum by an enzyme-linked immunosorbent assay (ELISA). A significant increase in the level of lipid peroxidation was found in SLE and RA patients and positively associated with disease activity. The activities of anti-oxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and anti-oxidant molecule GSH were significantly reduced in both diseases. Strong positive associations were found between MDA with RANTES/CCL5 and MIP-1β/CCL4 than MCP-1/CCL-2 in SLE patients while a sturdy connotation was seen with MIP-1β/CCL4 and MCP-1/CCL-2 in RA patients. The anti-oxidant molecule GSH shows a negative association with serum levels of MCP-1/CCL-2, RANTES/CCL5 and IP-10/CXCL-10 in SLE patients and with MCP-1/CCL-2 and RANTES/CCL5 in RA patients. A low level of GSH and high level of RANTES/CCL5 were associated with lupus nephritis patients. These results indicates that excessive production of ROS disturbs redox status and can modulate the expression of inflammatory chemokines leading to inflammatory processes, exacerbating inflammation and affecting tissue damage in autoimmune diseases, as exemplified by their strong association with disease activity.

Distinct phylogenetic lineages of Mycobacterium tuberculosis (MTB) cause disease in patients of particular genetic ancestry, and elicit different patterns of cytokine and chemokine secretion when cultured with human macrophages in vitro. Circulating and antigen-stimulated concentrations of these inflammatory mediators might therefore be expected to vary significantly between tuberculosis patients of different ethnic origin. Studies to characterise such variation, and to determine whether it relates to host or bacillary factors, have not been conducted. We therefore compared circulating and antigen-stimulated concentrations of 43 inflammatory mediators and 14 haematological parameters (inflammatory profile) in 45 pulmonary tuberculosis patients of African ancestry vs. 83 patients of Eurasian ancestry in London, UK, and investigated the influence of bacillary and host genotype on these profiles. Despite having similar demographic and clinical characteristics, patients of differing ancestry exhibited distinct inflammatory profiles at presentation: those of African ancestry had lower neutrophil counts, lower serum concentrations of CCL2, CCL11 and vitamin D binding protein (DBP) but higher serum CCL5 concentrations and higher antigen-stimulated IL-1 receptor antagonist and IL-12 secretion. These differences associated with ethnic variation in host DBP genotype, but not with ethnic variation in MTB strain. Ethnic differences in inflammatory profile became more marked following initiation of antimicrobial therapy, and immunological correlates of speed of elimination of MTB from the sputum differed between patients of African vs. Eurasian ancestry. Our study demonstrates a hitherto unappreciated degree of ethnic heterogeneity in inflammatory profile in tuberculosis patients that associates primarily with ethnic variation in host, rather than bacillary, genotype. Candidate immunodiagnostics and immunological biomarkers of response to antimicrobial therapy should be derived and validated in tuberculosis patients of different ethnic origin.

The CC-chemokine RANTES (regulated on activation normal T-cell expressed and secreted; CCL5) transduces multiple intracellular signals. Like all chemokines, it stimulates G protein-coupled receptor (GPCR) activity through interaction with its cognate chemokine receptor(s), but in addition also activates a GPCR-independent signaling pathway. Here, we show that the latter pathway is mediated by an interaction between RANTES and glycosaminoglycan chains of CD44. We provide evidence that this association, at both low, physiologically relevant, and higher, probably supraphysiologic concentrations of RANTES, induces the formation of a signaling complex composed of CD44, src kinases, and adapter molecules. This triggers the activation of the p44/42 mitogen-activated protein kinase (MAPK) pathway. By specifically reducing CD44 expression using RNA interference we were able to demonstrate that the p44/p42 MAPK activation by RANTES requires a high level of CD44 expression. As well as potently inhibiting the entry of CCR5 using HIV-1 strains, RANTES can enhance HIV-1 infectivity under certain experimental conditions. This enhancement process depends in part on the activation of p44/p42 MAPK. Here we show that silencing of CD44 in HeLa-CD4 cells prevents the activation of p44/p42 MAPK and leads to a substantial reduction in HIV-1 infectivity enhancement by RANTES.

Distinct subsets of dendritic cells (DCs) are present in blood, probably "en route" to different tissues. We have investigated the chemokines and adhesion molecules involved in the migration of myeloid (CD11c(+)) and plasmacytoid (CD123(+)) human peripheral blood DCs across vascular endothelium. Among blood DCs, the CD11c(+) subset vigorously migrated across endothelium in the absence of any chemotactic stimuli, whereas spontaneous migration of CD123(+) DCs was limited. In bare cell migration assays, myeloid DCs responded with great potency to several inflammatory and homeostatic chemokines, whereas plasmacytoid DCs responded poorly to all chemokines tested. In contrast, the presence of endothelium greatly favored transmigration of plasmacytoid DCs in response to CXCL12 (stromal cell-derived factor-1) and CCL5 (regulated on activation, normal T expressed and secreted). Myeloid DCs exhibited a very potent transendothelial migration in response to CXCL12, CCL5, and CCL2 (monocyte chemoattractant protein-1). Furthermore, we explored whether blood DCs acutely switch their pattern of migration to the lymph node-derived chemokine CCL21 (secondary lymphoid-tissue chemokine) in response to microbial stimuli [viral double-stranded (ds)RNA or bacterial CpG-DNA]. A synthetic dsRNA rapidly enhanced the response of CD11c(+) DCs to CCL21, whereas a longer stimulation with CpG-DNA was needed to trigger CD123(+) DCs responsive to CCL21. Use of blocking monoclonal antibodies to adhesion molecules revealed that both DC subsets used platelet endothelial cell adhesion molecule-1 to move across activated endothelium. CD123(+) DCs required beta(2) and beta(1) integrins to transmigrate, whereas CD11c(+) DCs may use integrin-independent mechanisms to migrate across activated endothelium.

OBJECTIVE

Mouse aorta smooth muscle cells (SMC) express tumor necrosis factor receptor superfamily member 1A (TNFR-1) and lymphotoxin beta-receptor (LTbetaR). Circumstantial evidence has linked the SMC LTbetaR to tertiary lymphoid organogenesis in hyperlipidemic mice. Here, we explored TNFR-1 and LTbetaR signaling in cultured SMC.

RESULTS

TNFR-1 signaling activated the classical RelA NF-kappaB pathway, whereas LTbetaR signaling activated the classical RelA and alternative RelB NF-kappaB pathways, and both signaling pathways synergized to enhance p100 inhibitor processing to the p52 subunit of NF-kappaB. Microarrays showed that simultaneous TNFR-1/LTbetaR activation resulted in elevated mRNA encoding leukocyte homeostatic chemokines CCL2, CCL5, CXCL1, and CX3CL1. Importantly, SMC acquired features of lymphoid tissue organizers, which control tertiary lymphoid organogenesis in autoimmune diseases through hyperinduction of CCL7, CCL9, CXCL13, CCL19, CXCL16, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. TNFR-1/LTbetaR cross-talk resulted in augmented secretion of lymphorganogenic chemokine proteins. Supernatants of TNFR-1/LTbetaR-activated SMC markedly supported migration of splenic T cells, B cells, and macrophages/dendritic cells. Experiments with ltbr(-/-) SMC indicated that LTbetaR-RelB activation was obligatory to generate the lymphoid tissue organizer phenotype.

CONCLUSIONS

SMC may participate in the formation of tertiary lymphoid tissue in atherosclerosis by upregulation of lymphorganogenic chemokines involved in T-lymphocyte, B-lymphocyte, and macrophage/dendritic cell attraction.

BACKGROUND

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized pathologically by the presence in the brain of intracellular protein inclusions highly enriched in aggregated alpha-synuclein (α-Syn). Although it has been established that progression of the disease is accompanied by sustained activation of microglia, the underlying molecules and factors involved in these immune-triggered mechanisms remain largely unexplored. Lately, accumulating evidence has shown the presence of extracellular α-Syn both in its aggregated and monomeric forms in cerebrospinal fluid and blood plasma. However, the effect of extracellular α-Syn on cellular activation and immune mediators, as well as the impact of familial PD-linked α-Syn mutants on this stimulation, are still largely unknown.

RESULTS

In this work, we have compared the activation profiles of non-aggregated, extracellular wild-type and PD-linked mutant α-Syn variants on primary glial and microglial cell cultures. After stimulation of cells with α-Syn, we measured the release of Th1- and Th2- type cytokines as well as IP-10/CXCL10, RANTES/CCL5, MCP-1/CCL2 and MIP-1α/CCL3 chemokines. Contrary to what had been observed using cell lines or for the case of aggregated α-Syn, we found strong differences in the immune response generated by wild-type α-Syn and the familial PD mutants (A30P, E46K and A53T).

CONCLUSIONS

These findings might contribute to explain the differences in the onset and progression of this highly debilitating disease, which could be of value in the development of rational approaches towards effective control of immune responses that are associated with PD.

A number of human immunodeficiency virus type-1 (HIV) positive subjects are also opiate abusers. These individuals are at high risk to develop neurological complications. However, little is still known about the molecular mechanism(s) linking opiates and HIV neurotoxicity. To learn more, we exposed rat neuronal/glial cultures prepared from different brain areas to opiate agonists and HIV envelope glycoproteins gp120IIIB or BaL. These strains bind to CXCR4 and CCR5 chemokine receptors, respectively, and promote neuronal death. Morphine did not synergize the toxic effect of gp120IIIB but inhibited the cytotoxic property of gp120BaL. This effect was blocked by naloxone and reproduced by the mu opioid receptor agonist DAMGO. To examine the potential mechanism(s) of neuroprotection, we determined the effect of morphine on the release of chemokines CCL5 and CXCL12 in neurons, astrocytes, and microglia cultures. CCL5 has been shown to prevent gp120BaL neurotoxicity while CXCL12 decreases neuronal survival. Morphine elicited a time-dependent release of CCL5 but failed to affect the release of CXCL12. This effect was observed only in primary cultures of astrocytes. To examine the role of endogenous CCL5 in the neuroprotective activity of morphine, mixed cerebellar neurons/glial cells were immunoneutralized against CCL5 prior to morphine and gp120 treatment. In these cells the neuroprotective effect of opiate agonists was blocked. Our data suggest that morphine may exhibit a neuroprotective activity against M-tropic gp120 through the release of CCL5 from astrocytes.

OBJECTIVE

Cardiovascular events remain the leading cause of death in rheumatoid arthritis (RA). To study the role of cytokines in these observations, the effects of tumour necrosis factor α (TNFα) and interleukin (IL)-17, a classical and a new key player in RA, were assessed in endothelial cell (EC) dysfunction.

METHODS

Primary human EC were treated with IL-17 alone or combined with TNFα. mRNA expression was quantified by qRT PCR and Affymetrix microarrays. The role of IL-17 was studied using functional assays of platelet aggregation, EC migration and invasion.

RESULTS

IL-17 alone induced 248 pro-inflammatory genes and 9803, when combined with TNFα. IL-17 plus TNFα induced synergistically chemokine genes such as CCL5, IL-8 and cytokine genes such as IL-6. In contrast, IL-17 decreased genes involved in the regulation of inflammation such as IL-33. IL-17 induced EC migration and invasion in synergy with TNFα. Such invasion was inhibited with an antiCXCR4 antibody, indicating the contribution of the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 axis. Supernatants of IL-17-treated EC induced strong platelet aggregation. IL-17 inhibited endothelial CD39/ATPDase expression, an inhibitor of platelet activation. Finally, IL-17 enhanced genes critical for coagulation such as tissue factor and decreased thrombomodulin, leading to a pro-thrombotic state.

CONCLUSIONS

These results indicate that IL-17 specifically when combined with TNFα has major pro-coagulant and pro-thrombotic effects on vessels.

BACKGROUND

Congenital human cytomegalovirus (HCMV) infections can result in CNS abnormalities in newborn babies including vision loss, mental retardation, motor deficits, seizures, and hearing loss. Brain pericytes play an essential role in the development and function of the blood-brain barrier yet their unique role in HCMV dissemination and neuropathlogy has not been reported.

METHODS

Primary human brain vascular pericytes were exposed to a primary clinical isolate of HCMV designated 'SBCMV'. Infectivity was analyzed by microscopy, immunofluorescence, Western blot, and qRT-PCR. Microarrays were performed to identify proinflammatory cytokines upregulated after SBCMV exposure, and the results validated by real-time quantitative polymerase chain reaction (qPCR) methodology. In situ cytokine expression of pericytes after exposure to HCMV was examined by ELISA and in vivo evidence of HCMV infection of brain pericytes was shown by dual-labeled immunohistochemistry.

RESULTS

HCMV-infected human brain vascular pericytes as evidenced by several markers. Using a clinical isolate of HCMV (SBCMV), microscopy of infected pericytes showed virion production and typical cytomegalic cytopathology. This finding was confirmed by the expression of major immediate early and late virion proteins and by the presence of HCMV mRNA. Brain pericytes were fully permissive for CMV lytic replication after 72 to 96 hours in culture compared to human astrocytes or human brain microvascular endothelial cells (BMVEC). However, temporal transcriptional expression of pp65 virion protein after SBCMV infection was lower than that seen with the HCMV Towne laboratory strain. Using RT-PCR and dual-labeled immunofluorescence, proinflammatory cytokines CXCL8/IL-8, CXCL11/ITAC, and CCL5/Rantes were upregulated in SBCMV-infected cells, as were tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), and interleukin-6 (IL-6). Pericytes exposed to SBCMV elicited higher levels of IL-6 compared to both mock-infected as well as heat-killed virus controls. A 6.6-fold induction of IL-6 and no induction TNF-alpha was observed in SBCMV-infected cell supernatants at 24 hours postinfection. Using archival brain tissue from a patient coinfected with HCMV and HIV, we also found evidence of HCMV infection of pericytes using dual-label immunohistochemistry, as monitored by NG2 proteoglycan staining.

CONCLUSIONS

HCMV lytic infection of primary human brain pericytes suggests that pericytes contribute to both virus dissemination in the CNS as well as neuroinflammation.

The expression of CCL5/Rantes by Hodgkin (H) and Reed-Sternberg (RS) cells has been recently documented. In the present study we demonstrated that the CCL5 receptor (CCR5) is constitutively expressed by Hodgkin Lymphoma (HL)-derived cell lines (i.e. L-428, KM-H2, L-1236 and L-540) as shown by immunohistochemistry, flow cytometry and western blotting and also detected by immunohistochemistry on primary H-RS cells from lymph node tissues. sCD40L never significantly affected CCR5 expression, whereas a short exposure to doxorubicin down regulated its expression. CCR5 receptors on HL cell lines were functionally active, since neutralizing anti-CCL5 monoclonal antibodies inhibited basal proliferation of HL-derived cell lines and recombinant CCR5 ligands (CCL3/Mip-1 alpha, CCL4/Mip1 beta and CCL5/Rantes) increased their clonogenic growth. CCL5 secretion by L-1236, L-428 and KM-H2 cells was stimulated by CD40 engagement and also by coculturing L-1236 cells on primary stromal fibroblasts from HL-involved lymph nodes (HLF). Coculture experiments indicated that a direct contact of H-RS cells induces HLF cells to produce CCL5. Supernatants from L-1236, L-428 and KM-H2 cells stimulated migration of purified CD4+ T-cells and eosinophils in vitro. The migratory response to HL-cell lines supernatants was only partially neutralized (CD4+ cells: 70%; esinophils: 36%) by anti-CCL5 antibodies, reinforcing the notion that multiple chemokines are involved in the recruitment of nonmalignant reactive cells in HL tissues. Taken together, our results indicate a possible involvement of the CCR5/CCR5-ligands signaling in the regulation of H-RS cells growth and in the formation/maintenance of the typical tissue microenvironment of HL.

Control of M.tb, the causative agent of TB, requires immune cell recruitment to form lung granulomas. The chemokines and chemokine receptors that promote cell migration for granuloma formation, however, are not defined completely. As immunity to M.tb manifests slowly in the lungs, a better understanding of specific roles for chemokines, in particular those that promote M.tb-protective T(H)1 responses, may identify targets that could accelerate granuloma formation. The chemokine CCL5 has been detected in patients with TB and implicated in control of M.tb infection. To define a role for CCL5 in vivo during M.tb infection, CCL5 KO mice were infected with a low dose of aerosolized M.tb. During early M.tb infection, CCL5 KO mice localized fewer APCs and chemokine receptor-positive T cells to the lungs and had microscopic evidence of altered cell trafficking to M.tb granulomas. Early acquired immunity and granuloma function were transiently impaired when CCL5 was absent, evident by delayed IFN-gamma responses and poor control of M.tb growth. Lung cells from M.tb-infected CCL5 KO mice eventually reached or exceeded the levels of WT mice, likely as a result of partial compensation by the CCL5-related ligand, CCL4, and not because of CCL3. Finally, our results suggest that most T cells use CCR5 but not CCR1 to interact with these ligands. Overall, these results contribute to a model of M.tb granuloma formation dependent on temporal regulation of chemokines rather than on redundant or promiscuous interactions.

CCL5 (RANTES) is a proinflammatory chemokine known to activate leukocytes through its receptor, CCR5. Although the monomeric form of CCL5 is sufficient to cause cell migration in vitro, CCL5's propensity for aggregation is essential for migration in vivo, T cell activation and apoptosis, and HIV entry into cells. However, there is currently no structural information on CCL5 oligomers larger than the canonical CC chemokine dimer. In this study the solution structure of a CCL5 oligomer was investigated using an integrated approach, including NMR residual dipolar couplings to determine allowed relative orientations of the component monomers, SAXS to restrict overall shape, and hydroxyl radical footprinting and NMR cross-saturation experiments to identify interface residues. The resulting model of the CCL5 oligomer provides a basis for explaining the disaggregating effect of E66 and E26 mutations and suggests mechanisms by which glycosaminoglycan binding may promote oligomer formation and facilitate cell migration in vivo.

RANTES (CCL5) is a chemokine expressed by many hematopoietic and non-hematopoietic cell types that plays an important role in homing and migration of effector and memory T cells during acute infections. The RANTES receptor, CCR5, is a major target of anti-HIV drugs based on blocking viral entry. However, defects in RANTES or RANTES receptors including CCR5 can compromise immunity to acute infections in animal models and lead to more severe disease in humans infected with west Nile virus (WNV). In contrast, the role of the RANTES pathway in regulating T cell responses and immunity during chronic infection remains unclear. In this study, we demonstrate a crucial role for RANTES in the control of systemic chronic LCMV infection. In RANTES⁻/⁻ mice, virus-specific CD8 T cells had poor cytokine production. These RANTES⁻/⁻ CD8 T cells also expressed higher amounts of inhibitory receptors consistent with more severe exhaustion. Moreover, the cytotoxic ability of CD8 T cells from RANTES⁻/⁻ mice was reduced. Consequently, viral load was higher in the absence of RANTES. The dysfunction of T cells in the absence of RANTES was as severe as CD8 T cell responses generated in the absence of CD4 T cell help. Our results demonstrate an important role for RANTES in sustaining CD8 T cell responses during a systemic chronic viral infection.

OBJECTIVE

We investigated whether retinal ischemia and inflammation produced by raising the intraocular pressure above normal systolic levels differs in mice that lack a functional toll-like receptor 4 (Tlr4) signaling pathway.

METHODS

In this work we used the murine strain B6.B10ScN-Tlr4(lps-del)/JthJ, which does not express functional Tlr4. C57BL/6J was considered as the control. We induced retinal ischemia by unilateral elevation of intraocular pressure for 1 h by direct corneal cannulation. The changes in expression of proinflammatory genes 24 h postreperfusion were assessed by quantitative PCR. Corresponding changes in protein abundances were analyzed by western blot and immunohistochemistry. Cell death was evaluated by direct counting of neurons in the ganglion cell layer of flat-mounted retinas seven days postreperfusion.

RESULTS

We showed that Tlr4-deficient mice display significantly reduced expression of proinflammatory genes, including RelA, tumor necrosis factor (Thf), interleukin 6 (Il6), chemokine (C-C motif) ligand 2 (Ccl2), chemokine (C-C motif) ligand 5 (Ccl5), chemokine (C-X-C motif) ligand 10 (Cxcl10), Cybb, nitric oxide synthase 2 (Nos2), and intercellular adhesion molecule 1 (Icam1) 24 h after reperfusion. The mice that lacked Tlr4 showed significantly increased survival of neurons in the ganglion cell layer following ischemic injury, as compared to wild-type controls.

CONCLUSIONS

Our results indicate that Tlr4 signaling is involved in retinal damage and inflammation triggered by ischemic injury.