Macrophages activate the production of cytokines and chemokines in response to LPS through signaling cascades downstream from TLR4. Lipid mediators such as PGE(2), which are produced during inflammatory responses, have been shown to suppress MyD88-dependent gene expression upon TLR4 activation in macrophages. The study reported here investigated the effect of PGE(2) on TLR3- and TLR4-dependent, MyD88-independent gene expression in murine J774A.1 macrophages, as well as the molecular mechanism underlying such an effect. We demonstrate that PGE(2) strongly suppresses LPS-induced IFN-beta production at the mRNA and protein levels. Poly (I:C)-induced IFN-beta and LPS-induced CCL5 production were also suppressed by PGE(2). The inhibitory effect of PGE(2) on LPS-induced IFN-beta expression is mediated through PGE(2) receptor subtypes EP(2) and EP(4), and mimicked by the cAMP analog 8-Br-cAMP as well as by the adenylyl cyclase activator forskolin. The downstream effector molecule responsible for the cAMP-induced suppressive effect is exchange protein directly activated by cAMP (Epac) but not protein kinase A. Moreover, data demonstrate that Epac-mediated signaling proceeds through PI3K, Akt, and GSK3beta. In contrast, PGE(2) inhibits LPS-induced TNF-alpha production in these cells through a distinct pathway requiring protein kinase A activity and independent of Epac/PI3K/Akt. In vivo, administration of a cyclooxygenase inhibitor before LPS injection resulted in enhanced serum IFN-beta concentration in mice. Collectively, data demonstrate that PGE(2) is a negative regulator for IFN-beta production in activated macrophages and during endotoxemia.

Despite accumulating evidence, the role of leptin in chemokine expression is poorly understood. In this study, we evaluated the effects of leptin on CC-chemokine ligands (CCLs), CCL3, CCL4, and CCL5 gene expression in cultured murine macrophage, J774A.1 cells. Expression of all these CCLs mRNA was gradually increased and significant up-regulation was observed for 3-12 h exposure to leptin (1 microM). The phosphorylated signal transducer and activator of transcription 3 (pSTAT3) was significantly increased for 5-20 min exposure to leptin, and it was localized in leptin receptor-positive macrophage. Pretreatment with AG490 (100 microM), a janus kinase 2 (JAK2) inhibitor, significantly suppressed leptin-induced pSTAT3 increases and the up-regulation of CCLs mRNA expression. In conclusion, leptin enhances CCLs expression in cultured murine macrophage, through activation of a JAK2-STAT3 pathway. Therefore, a new paradigm of leptin-mediated chemokine expression may lead to the clarification of complex immune systems in future.

BACKGROUND

Chronic skin inflammation in atopic dermatitis (AD) is associated with elevated expression of proinflammatory genes and activation of innate immune responses in keratinocytes. microRNAs (miRNAs) are short, single-stranded RNA molecules that silence genes via the degradation of target mRNAs or inhibition of translation.

OBJECTIVE

The aim of this study was to investigate the role of miR-146a in skin inflammation in AD.

METHODS

RNA and protein expression was analyzed using miRNA and mRNA arrays, RT-quantitative PCR, Western blotting, and immunonohistochemistry. Transfection of miR-146a precursors and inhibitors into human primary keratinocytes, luciferase assays, and MC903-dependent mouse model of AD were used to study miR-146a function.

RESULTS

We show that miR-146a expression is increased in keratinocytes and chronic lesional skin of patients with AD. miR-146a inhibited the expression of numerous proinflammatory factors, including IFN-γ-inducible and AD-associated genes CCL5, CCL8, and ubiquitin D (UBD) in human primary keratinocytes stimulated with IFN-γ, TNF-α, or IL-1β. In a mouse model of AD, miR-146a-deficient mice developed stronger inflammation characterized by increased accumulation of infiltrating cells in the dermis, elevated expression of IFN-γ, CCL5, CCL8, and UBD in the skin, and IFN-γ, IL-1β, and UBD in draining lymph nodes. Both tissue culture and in vivo experiments in mice demonstrated that miR-146a-mediated suppression in allergic skin inflammation partially occurs through direct targeting of upstream nuclear factor kappa B signal transducers caspase recruitment domain-containing protein 10 and IL-1 receptor-associated kinase 1. In addition, human CCL5 was determined as a novel, direct target of miR-146a.

CONCLUSIONS

Our data demonstrate that miR-146a controls nuclear factor kappa B-dependent inflammatory responses in keratinocytes and chronic skin inflammation in AD.

Vascular access dysfunction compromises the care of patients on chronic hemodialysis. Elucidating the mechanisms of such dysfunction and devising strategies that may interrupt neointimal hyperplasia and relevant pathogenetic pathways are essential. Here, we show that, in the venous segment of a murine model of an arteriovenous fistula, monocyte chemoattractant protein-1 (MCP-1) mRNA and protein increase, accompanied by increased activity of the transcription factors NF-κB and AP-1. Genetic deficiency of MCP-1 proved markedly protective in this murine model, reflected by increased fistula patency 6 weeks after its formation, decreased venous wall thickness, and increased luminal area. An early effect of MCP-1 deficiency was the attenuation of the marked induction of CCL5 (RANTES) that occurred in this model, a chemokine recently recognized as a critical participant in vascular injury. Finally, in a rat model of an arteriovenous fistula, we localized expression of MCP-1 to the endothelium, proliferating smooth muscle cells and infiltrating leukocytes. In summary, marked upregulation of MCP-1 occurs in the venous segment of an arteriovenous fistula in rodents, and this vasculopathic chemokine contributes to failure of the fistula.

Age-related macular degeneration (AMD) is the leading cause of blindness in the United States. Ccl2 knock-out (KO) mice sporadically develop the cardinal features of AMD in their senescent stage. Humans bearing a loss of function variant or single nucleotide polymorphism (SNP) in CX3CR1 are at increased risk of developing AMD. We recently developed Ccl2(-/-)/Cx3cr1(-/-) mice, which consistently develop retinal degeneration with many AMD features. Since there is strong evidence for an immunological role in AMD pathogenesis, we examined ocular immune protein expression levels in Ccl2(-/-)/Cx3cr1(-/-), Ccl2(-/-), Cx3cr1(-/-), and age-matched wild-type (WT) mice. Immunohistochemistry revealed increased complement C3d in Bruch's membrane, retinal pigment epithelium (RPE), choroidal capillaries, and particularly drusen of the Ccl2(-/-)/Cx3cr1(-/-) mice relative to the WT controls. No change was detected in single KO mice. Real-time RT-PCR revealed a 2.5-fold increase in C3 expression in the Ccl2(-/-)/Cx3cr1(-/-). While the retinas of four month old WT and Ccl2(-/-) showed minimal immunoreactivity for markers of macrophages and microglia, infiltrates of these mononuclear phagocytic cells were detected in the Ccl2(-/-)/Cx3cr1(-/-)retinal lesions and a few foci in the Cx3cr1(-/-) retina. The Ccl2(-/-)/Cx3cr1(-/-) had reduced toll-like receptor 4 (TLR4) expression in the RPE. Following LPS injection, the Ccl2(-/-)/Cx3cr1(-/-) had significantly reduced endotoxin-induced uveitis scores and showed a diminished increase in Tlr4 mRNA expression. No changes in TLR4 expression were detected in either single KO. Autoantibodies against the retina and photoreceptors were also detected in the Ccl2(-/-)/Cx3cr1(-/-) serum. Real-time RT-PCR revealed significant increases in Ccl5 transcript in the Ccl2(-/-)/Cx3cr1(-/-) relative to the WT. These results suggest that innate immunity and possibly adaptive immunity play an important role in Ccl2(-/-)/Cx3cr1(-/-) retinal degeneration. Moreover, since human AMD patients show similar immunopathological profiles, these results support the Ccl2(-/-)/Cx3cr1(-/-) as a suitable model for human AMD.

Natural killer (NK) cells comprise a set of lymphocytes that is capable of mediating innate immune responses to viral infections, malignancies, and allogeneic bone marrow grafts. This review summarizes what is known about the mechanisms NK cells use to arrive at their sites of action. NK cells express a wide array of adhesion molecules including alphaLbeta2, alphaMbeta2, alphaXbeta2, and alpha4beta1 integrins, ICAM-1, PSGL-1, and L-selectin. Like other immune and inflammatory cells, NK cells use the blood circulation to enter tissues and organs, which requires that they interact with the vessel wall under flow conditions, arrest, and transmigrate. NK cells are able to chemotax to a variety of cytokines and chemokines, including IL-12, IFN-(alpha/beta, CCL2, 3, 4, 5, 7, 8, CXCL8, and CX3CL1. In many cases, NK cells appear to migrate towards these soluble factors without any kind of priming. These cells also appear to distribute in secondary and tertiary lymphoid sites (i.e., spleen, bone marrow, liver, lung, and lymph nodes) both with and without stimulation. In addition to their ability to move throughout the body in an unprimed state, activated NK cells may have increased specificity in homing to sites of inflammation. NK cells not only react to, but also produce IFN-gamma, TNF-alpha, GM-CSF, CCL3, CCL4, and CCL5, enabling them to recruit various immune cells to sites of immune response.

BACKGROUND

Increased numbers of activated neutrophils have been reported in the bronchial mucosa of patients with stable chronic obstructive pulmonary disease (COPD), particularly in severe disease.

OBJECTIVE

To investigate the expression of neutrophilic chemokines and adhesion molecules in bronchial biopsies from patients with stable COPD of different severity (GOLD stages I-IV) compared with age-matched control subjects, smokers with normal lung function and never smokers.

METHODS

The expression of CCL5, CXCL1, 5, 6, 7 and 8, CXCR1, CXCR2, CD11b and CD44 was measured in the bronchial mucosa using immunohistochemistry, confocal immunofluorescence, real-time quantitative polymerase chain reaction (RT-QPCR) and Western blotting (WB).

RESULTS

The numbers of CCL5+ epithelial cells and CCL5+ and CXCL7+ immunostained cells were increased in the bronchial submucosa of patients with stable severe COPD compared with control never smokers and smokers with normal lung function. This was also confirmed at the level of mRNA expression. The numbers of CCL5+ cells in the submucosa of patients with COPD were 2-15 times higher than any other chemokines. There was no correlation between the number of these cells and the number of neutrophils in the bronchial submucosa. Compared with control smokers, the percentage of neutrophils co-expressing CD11b and CD44 receptors was significantly increased in the submucosa of patients with COPD.

CONCLUSIONS

The increased expression of CCL5 and CXCL7 in the bronchial mucosa of patients with stable COPD, together with an increased expression of extracellular matrix-binding receptors on neutrophils, may be involved in the pathogenesis of COPD.

BACKGROUND

Chronic Chagas cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi. Even though the Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis, little is known about the factors controlling inflammatory cell migration to CCC myocardium.

RESULTS

Using confocal immunofluorescence and quantitative PCR, we studied cell surface staining and gene expression of the CXCR3, CCR4, CCR5, CCR7, CCR8 receptors and their chemokine ligands in myocardial samples from end-stage CCC patients. CCR5+, CXCR3+, CCR4+, CCL5+ and CXCL9+ mononuclear cells were observed in CCC myocardium. mRNA expression of the chemokines CCL5, CXCL9, CXCL10, CCL17, CCL19 and their receptors was upregulated in CCC myocardium. CXCL9 mRNA expression directly correlated with the intensity of myocarditis, as well as with mRNA expression of CXCR3, CCR4, CCR5, CCR7, CCR8 and their ligands. We also analyzed single-nucleotide polymorphisms for genes encoding the most highly expressed chemokines and receptors in a cohort of Chagas disease patients. CCC patients with ventricular dysfunction displayed reduced genotypic frequencies of CXCL9 rs10336 CC, CXCL10 rs3921 GG, and increased CCR5 rs1799988CC as compared to those without dysfunction. Significantly, myocardial samples from CCC patients carrying the CXCL9/CXCL10 genotypes associated to a lower risk displayed a 2-6 fold reduction in mRNA expression of CXCL9, CXCL10, and other chemokines and receptors, along with reduced intensity of myocarditis, as compared to those with other CXCL9/CXCL10 genotypes.

CONCLUSIONS

Results may indicate that genotypes associated to reduced risk in closely linked CXCL9 and CXCL10 genes may modulate local expression of the chemokines themselves, and simultaneously affect myocardial expression of other key chemokines as well as intensity of myocarditis. Taken together our results may suggest that CXCL9 and CXCL10 are master regulators of myocardial inflammatory cell migration, perhaps affecting clinical progression to the life-threatening form of CCC.

The acquisition of a metastatic phenotype in breast epithelial cells is a progressive process, influenced by a large variety of cellular and soluble factors. Of these, members of the chemokine superfamily, such as CCL2, CCL5, CXCL8 and CXCL12 have been recently suggested to promote breast cancer progression. A pre-requisite for elucidation of the role of other chemokines in breast cancer progression is the characterization of chemokine and chemokine receptor expression by breast tumor cells. The present study focuses on CXCL10, a CXC chemokine that was recently suggested to have anti-malignant properties, and its corresponding receptor CXCR3. CXCR3 expression was detected in three human breast adenocarcinoma cell lines, MDA-MB-231, MCF-7 and T47D. CXCR3 expression was potently up-regulated by growing the cells under stress conditions, imposed by serum starvation. Unlike many other chemokine receptors, CXCR3 expression was not down-regulated by exposure to high concentrations (500ng/ml) of its ligand, CXCL10, but rather was promoted. CXCL10-induced up-regulation of CXCR3 expression in the three cell lines was inhibited by cycloheximide, indicating that de novo protein synthesis is required for this process. In addition to CXCR3, the secretion of CXCL10 was noted in the MDA-MB-231, MCF-7 and T47D cells. CXCL10 secretion was found to be down-regulated by IL-6, a potentially pro-malignant cytokine in breast cancer. The concomitant expression of CXCR3 and CXCL10 in breast tumor cells suggests that a CXCR3-CXCL10 axis may function in these cells, and paves the way for an in depth analysis of CXCL10-CXCR3 interactions in breast tumor cells.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial, rate-limiting step of tryptophan (Trp) catabolism along the kynurenine (KYN) pathway, and its induction in cells of the immune system in response to cytokines has been implicated in the regulation of antigen presentation and responses to cell-mediated immune attack. Microarray and quantitative PCR analyses of isolated human islets incubated with interferon (IFN)-gamma for 24 h revealed increased expression of IDO mRNA (>139-fold) and Trp-tRNA synthase (WARS) (>17-fold) along with 975 other transcripts more than threefold, notably the downstream effectors janus kinase (JAK)2, signal transducer and activator of transcription (STAT)1, IFN-gamma regulatory factor-1, and several chemokines (CXCL9/MIG, CXCL10/IP10, CXCL11/1-TAC, CCL2, and CCL5/RANTES) and their receptors. IDO protein expression was upregulated in IFN-gamma-treated islets and accompanied by increased intracellular IDO enzyme activity and the release of KYN into the media. The response to IFN-gamma was countered by interleukin-4 and 1alpha-methyl Trp. Immunohistochemical localization showed IDO to be induced in cells of both endocrine, including pancreatic duodenal homeobox 1-positive beta-cells, and nonendocrine origin. We postulate that in the short term, IDO activation may protect islets from cytotoxic damage, although chronic exposure to various Trp metabolites could equally lead to beta-cell attrition.

HL is a malignant lymphoma characterized by a small number of malignant HRS cells among a major population of infiltrating reactive cells, e.g., lymphocytes and eosinophils. We previously reported that mast cells are present in HL-affected lymph nodes and therein are the predominant CD30L-expressing cells. The CD30L expressed on mast cells is functionally active and can provide stimulatory signals to HRS cells. Thus, mast cells constitute an important portion of the infiltrating reactive cells that contribute to tumor progression in HL. Control of the recruitment of this previously unrecognized cell and its interactions with tumor cells are essentially unknown. To elucidate if mast cells might be specifically attracted to the tumor area by chemokines produced by HRS cells, we investigated chemokine expression in HL cell lines and in vivo. By RNase protection assay, mRNA expression of several chemokines could be detected in the cell lines. Despite the heterogeneous expression profile exhibited by the cell lines, 4 of 5 expressed CCL5 (RANTES) mRNA. RT-PCR and immunohistochemistry confirmed expression of CCL5 in vivo. Furthermore, secreted CCL5 was detected in conditioned media from 3 of the cell lines. In a migration assay, we found that CCL5 present in conditioned medium could induce mast cell migration. Taken together, our results suggest that CCL5 produced by HRS cells is one mechanism by which mast cells can be attracted into the tumor tissue in HL.

Circulating bone marrow-derived Mesenchymal Stem Cells (BM-MSCs) have an innate tropism for tumor tissue in response to the inflammatory microenvironment present in malignant lesions. The prostate is bombarded by numerous infectious and inflammatory insults over a lifetime. Chronic inflammation is associated with CXCL12, CCL5, and CCL2, which are highly overexpressed in prostate cancer. Among other cell types, these chemoattractant stimuli recruit BM-MSCs to the tumor. MSCs are minimally defined as plastic-adhering cells characterized by the expression of CD90, CD73, and CD105 in the absence of hematopoietic markers, which can differentiate into osteoblasts, chondrocytes, and adipocytes. MSCs are immunoprivileged and have been implicated in tumorigenesis through multiple mechanisms, including promoting proliferation, angiogenesis, and metastasis, in addition to the generation of an immunosuppressive microenvironment. We have demonstrated that MSCs represent 0.01-1.1% of the total cells present in core biopsies from primary human prostatectomies. Importantly, these analyses were performed on samples prior to expansion in tissue culture. MSCs in these prostatectomy samples are FAP-, CD90-, CD73-, and CD105-positive, and CD14-, CD20-, CD34-, CD45-, and HLA-DR-negative. Additionally, like BM-MSCs, these prostate cancer-derived stromal cells (PrCSCs) were shown to differentiate into osteoblasts, adipocytes and chondrocytes. In contrast to primary prostate cancer-derived epithelial cells, fluorescently-labeled PrCSCs and BM-MSCs were both shown to home to CWR22RH prostate cancer xenografts following IV injection. These studies demonstrate that not only are MSCs present in sites of prostate cancer where they may contribute to carcinogenesis, but these cells may also potentially be used to deliver cytotoxic or imaging agents for therapeutic and/or diagnostic purposes.

Impairment in two blood-brain barrier (BBB) efflux transporters, p-glycoprotein (Pgp) and low-density lipoprotein receptor-related protein-1 (LRP-1) are thought to contribute to the progression of Alzheimer's disease (AD) by resulting in the brain accumulation of their substrate amyloid beta peptide (Aβ). The initial cause of impaired efflux, however, is unknown. We have shown that induction of systemic inflammation by intraperitoneal administration of lipopolysaccharide impairs the efflux of Aβ from the brain, suggesting that systemic inflammation could be one such initiator. In this study, we determined whether pre-administration of the antioxidant N-aceytlcysteine (Nac) has a protective effect against LPS-induced Aβ transporter dysfunction. Our findings were that Nac protected against LPS-induced Aβ transport dysfunction at the BBB through an LRP-1-dependent and Pgp-independent mechanism. This was associated with Nac exerting antioxidant effects in the periphery but not the brain, despite an increased rate of entry of Nac into the brain following LPS. We also found that Nac pre-administration resulted in lower blood levels of the cytokines and chemokines interferon-γ, interleukin-10, CCL2, CCL4, and CCL5, but only lowered CCL4 in the cerebral cortex and hippocampus. Finally, we observed that hippocampal cytokine responses to LPS were decreased compared to cortex. These findings demonstrate a novel mechanism by which antioxidants prevent Aβ accumulation in the brain caused by inflammation, and therefore protect against AD.

The tumor microenvironment (TME) exerts critical pro-tumorigenic effects through cytokines and growth factors that support cancer cell proliferation, survival, motility and invasion. Insulin-like growth factor-1 (IGF-1) and signal transducer and activator of transcription 3 (STAT3) stimulate colorectal cancer development and progression via cell autonomous and microenvironmental effects. Using a unique inhibitor, NT157, which targets both IGF-1 receptor (IGF-1R) and STAT3, we show that these pathways regulate many TME functions associated with sporadic colonic tumorigenesis in CPC-APC mice, in which cancer development is driven by loss of the Apc tumor suppressor gene. NT157 causes a substantial reduction in tumor burden by affecting cancer cells, cancer-associated fibroblasts (CAF) and myeloid cells. Decreased cancer cell proliferation and increased apoptosis were accompanied by inhibition of CAF activation and decreased inflammation. Furthermore, NT157 inhibited expression of pro-tumorigenic cytokines, chemokines and growth factors, including IL-6, IL-11 and IL-23 as well as CCL2, CCL5, CXCL7, CXCL5, ICAM1 and TGFβ; decreased cancer cell migratory activity and reduced their proliferation in the liver. NT157 represents a new class of anti-cancer drugs that affect both the malignant cell and its supportive microenvironment.

Chemokine receptors belong to the family of G-protein-coupled receptors (GPCR). Phosphorylation of GPCR by GPCR kinases (GRKs) is considered to play an important role in desensitization of these receptors. We have recently shown in patients with rheumatoid arthritis that the level of GRK2 in lymphocytes is reduced by approximately 50%. However, the physiological relevance of reduced GRK2 levels in lymphocytes is not known. Here, we investigated whether reduced GRK2 expression changes the chemotactic response of T cells to the chemokines CCL3, CCL4, and CCL5. Activated T cells from GRK2+/- mice, which have a 50% reduction in GRK2 protein levels, showed a significant 40% increase in chemotaxis toward the CCR5 ligand CCL4. In addition, chemotaxis toward the CCR1 and CCR5 ligands CCL3 and CCL5 was also increased. Binding of CCL4 to activated T cells from GRK2+/- and wild-type (WT) mice was similar, but agonist-induced CCR5 phosphorylation was attenuated in GRK2+/- cells. Moreover, the calcium response and phosphorylation of protein kinase B and extracellular-regulated kinase in response to CCL4 were significantly increased in GRK2+/- T cells, showing that signaling is increased when the level of GRK2 is reduced. GRK2+/- and WT cells do become refractory to restimulation with CCL4. In conclusion, a 50% decrease in T cell GRK2 expression results in increased responsiveness to CCL3, CCL4, and CCL5, suggesting that the 50% reduction in lymphocyte GRK2 level as observed during inflammation can have functional consequences for the response of these cells to chemokines.

To glean biological differences and similarities of peripheral T-cell lymphoma-not otherwise specified [PTCL-NOS] to diffuse large B-cell lymphoma (DLBCL), a transcriptosome analysis was done on five PTCL-NOS and four DLBCL patients and validated by quantitative real-time reverse transcription-PCR on 10 selected genes. Normal peripheral blood T cells, peripheral blood B cells, and lymph node were used as controls. The resultant gene expression profile delineated distinct "tumor profile signatures" for PTCL-NOS and DLBCL. Several highly overexpressed genes in both PTCL-NOS and DLBCL involve the immune network, stroma, angiogenesis, and cell survival cascades that make important contributions to lymphomagenesis. Inflammatory chemokines and their receptors likely play a central role in these complex interrelated pathways: CCL2 and CXCR4 in PTCL-NOS and CCL5 and CCR1 in DLBCL. Highly overexpressed oncogenes unique to PTCL-NOS are SPI1, STK6, alpha-PDGFR, and SH2D1A, whereas in DLBCL they are PIM1, PIM2, LYN, BCL2A1, and RAB13. Oncogenes common to both lymphomas are MAFB, MET, NF-kappaB2, LCK, and LYN. Several tumor suppressors are also down-regulated (TPTE, MGC154, PTCH, ST5, and SUI1). This study illustrates the relevance of tumor-stroma immune trafficking and identified potential novel prognostic markers and targets for therapeutic intervention.

The substance P (SP)-preferring receptor neurokinin-1 receptor (NK-1R) has two forms: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. These two receptors differ in the length of the C terminus of NK-1R. We studied the undifferentiated and phorbol myristate acetate (PMA)-differentiated human monocyte/macrophage cell line THP-1 to investigate the expression and function of NK-1R. The expression of full-length and truncated NK-1R in this cell line was determined by using real-time PCR and immunofluorescence staining. Undifferentiated THP-1 cells expressed only truncated NK-1R. The differentiation of THP-1 cells with PMA to a macrophage-like phenotype resulted in the expression of full-length NK-1R, which was functionally accompanied by an SP (10(-6) M)-induced Ca2+ increase. In contrast, the addition of SP (10(-6) M) did not trigger Ca2+ response in undifferentiated THP-1 cells; however, SP did enhance the CCR5-preferring ligand RANTES (CCL5)-mediated Ca2+ increase. When a plasmid containing the full-length NK-1R was introduced into undifferentiated THP-1 cells, exposure to SP triggered Ca2+ increase, demonstrating that the full-length NK-1R is required for SP-induced Ca2+ increase. The NK-1R antagonist aprepitant (Emend, Merck) inhibited both the SP-induced Ca2+ increase in PMA-differentiated THP-1 cells and the SP priming effect on the CCL5-mediated Ca2+ increase, indicating that these effects are mediated through the full-length and truncated NK-1R, respectively. Taken together, these observations demonstrate that there are unique characteristics of NK-1R expression and NK-1R-mediated signaling between undifferentiated THP-1 cells and THP-1 cells differentiated to the macrophage phenotype.

To assess the role of CC-chemokine ligand 5 (CCL5)/RANTES in opiate drug abuse and human immunodeficiency virus type 1 (HIV-1) comorbidity, the effects of systemic morphine and intrastriatal HIV-1 Tat on macrophage/microglial and astroglial activation were assessed in wild-type and CCL5 knockout mice. Mice were injected intrastriatally with vehicle or Tat and assessed after 7 days. Morphine was administered to some Tat-injected mice via time-release implant (5 mg/day, s.c. for 5 days) starting at 2 days post injection. Glial activation was significantly reduced in CCL5(-/-) compared to wild-type mice at 7 days following combined Tat and morphine exposure. Moreover, the percentage of 3-nitrotyrosine immunopositive macrophages/microglia was markedly reduced in CCL5(-/-) mice injected with Tat +/- morphine compared to wild-type counterparts, suggesting that CCL5 contributes to nitrosative stress in HIV-1 encephalitis. In CCL5(-/-) mice, the reductions in Tat +/- morphine-induced gliosis coincided with significant declines in the proportion of CCL2/MCP-1-immunoreactive astrocytes and macrophages/microglia compared to wild-type counterparts. In knockout mice, neither Tat alone nor in combination with morphine increased the proportion of CCL2-immunoreactive astrocytes above percentages seen in vehicle-injected controls. Macrophages/microglia differed showing modest, albeit significant, increases in the proportion of CCL2-positive cells with combined Tat and morphine exposure, suggesting that CCL5 preferentially affects CCL2 expression by astroglia. Thus, CCL5 mediates glial activation caused by Tat and morphine, thereby aggravating HIV-1 neuropathogenesis in opiate abusers and non-abusers. CCL5 is implicated as mediating the cytokine-driven amplification of CCL2 production by astrocytes and resultant macrophage/microglial recruitment and activation.

CCL5 (RANTES (regulated on activation normal T cell expressed and secreted)) and its cognate receptor, CCR5, have been implicated in T cell activation. CCL5 binding to glycosaminoglycans (GAGs) on the cell surface or in extracellular matrix sequesters CCL5, thereby immobilizing CCL5 to provide the directional signal. In two CCR5-expressing human T cell lines, PM1.CCR5 and MOLT4.CCR5, and in human peripheral blood-derived T cells, micromolar concentrations of CCL5 induce apoptosis. CCL5-induced cell death involves the cytosolic release of cytochrome c, the activation of caspase-9 and caspase-3, and poly(ADP-ribose) polymerase cleavage. CCL5-induced apoptosis is CCR5-dependent, since native PM1 and MOLT4 cells lacking CCR5 expression are resistant to CCL5-induced cell death. Furthermore, we implicate tyrosine 339 as a critical residue involved in CCL5-induced apoptosis, since PM1 cells expressing a tyrosine mutant receptor, CCR5Y339F, do not undergo apoptosis. We show that CCL5-CCR5-mediated apoptosis is dependent on cell surface GAG binding. The addition of exogenous heparin and chondroitin sulfate and GAG digestion from the cell surface protect cells from apoptosis. Moreover, the non-GAG binding variant, (44AANA47)-CCL5, fails to induce apoptosis. To address the role of aggregation in CCL5-mediated apoptosis, nonaggregating CCL5 mutant E66S, which forms dimers, and E26A, which form tetramers at micromolar concentrations, were utilized. Unlike native CCL5, the E66S mutant fails to induce apoptosis, suggesting that tetramers are the minimal higher ordered CCL5 aggregates required for CCL5-induced apoptosis. Viewed altogether, these data suggest that CCL5-GAG binding and CCL5 aggregation are important for CCL5 activity in T cells, specifically in the context of CCR5-mediated apoptosis.

Information about the time and place of gene transcription, which until recently was only possible by extensive experimental analysis, can now be predicted through in silico analysis. Using the human RANTES/CCL5 promoter, we show that organizational features of promoters derived from promoter sequences contain information about the spatial and temporal 'functional context' of expression.

BACKGROUND

The highly prevalent obstructive sleep apnea syndrome (OSA) with its main component intermittent hypoxia (IH) is a risk factor for cardiovascular mortality. The poor knowledge of its pathophysiology has limited the development of specific treatments, whereas the gold standard treatment, continuous positive airway pressure, may not fully reverse the chronic consequences of OSA and has limited acceptance in some patients.

OBJECTIVE

To examine the contribution of IH-induced inflammation to the cardiovascular complications of OSA.

METHODS

We investigated systemic and vascular inflammatory changes in C57BL6 mice exposed to IH (21-5% Fi(O(2)), 60-s cycle) or normoxia 8 hours per day up to 14 days. Vascular alterations were reassessed in mice treated with a blocking antibody of regulated upon activation, normal T-cell expressed and secreted (RANTES)/CC chemokine ligand 5 (CCL5) signaling pathway, or with the IgG isotype control throughout the IH exposure.

RESULTS

IH induced systemic inflammation combining increased splenic lymphocyte proliferation and chemokine expression, with early and predominant RANTES/CCL5 alterations, and enhanced splenocyte migration toward RANTES/CCL5. IH also induced structural and inflammatory vascular alterations. Leukocyte-endothelium adhesive interactions were increased, attested by leukocyte rolling and intercellular adhesion molecule-1 expression in mesenteric vessels. Aortas had increased intima-media thickness with elastic fiber alterations, mucoid depositions, nuclear factor-κB-p50 and intercellular adhesion molecule-1 overexpression, hypertrophy of smooth-muscle cells overexpressing RANTES/CCL5, and adventitial-periadventitial T-lymphocyte infiltration. RANTES/CCL5 neutralization prevented both intima-media thickening and inflammatory alterations, independently of the IH-associated proatherogenic dyslipidemia.

CONCLUSIONS

Inflammation is a determinant mechanism for IH-induced preatherosclerotic remodeling involving RANTES/CCL5, a key chemokine in atherogenesis. Characterization of the inflammatory response could allow identifying at-risk patients for complications, and its pharmacologic manipulation may represent a potential complementary treatment of sleep apnea consequences.

The intraspinal cues that orchestrate T-cell migration and activation after spinal contusion injury were characterized using B10.PL (wild-type) and transgenic (Tg) mice with a T-cell repertoire biased toward recognition of myelin basic protein (MBP). Previously, we showed that these strains exhibit distinct anatomical and behavioral phenotypes. In Tg mice, MBP-reactive T-cells are activated by spinal cord injury (SCI), causing more severe axonal injury, demyelination, and functional impairment than is found in non-Tg wild-type mice (B10.PL). Conversely, despite a robust SCI-induced T-cell response in B10.PL mice, no overt T-cell-mediated pathology was evident. Here, we show that chronic intraspinal T-cell accumulation in B10.PL and Tg mice is associated with a dramatic and sustained increase in CXCL10/IP-10 and CCL5/RANTES mRNA expression. However, in Tg mice, chemokine mRNA were enhanced 2- to 17-fold higher than in B10.PL mice and were associated with accelerated intraspinal T-cell influx and enhanced CNS macrophage activation throughout the spinal cord. These data suggest common molecular pathways for initiating T-cell responses after SCI in mice; however, if T-cell reactions are biased against MBP, molecular and cellular determinants of neuroinflammation are magnified in parallel with exacerbation of neuropathology and functional impairment.

The involvement of macrophages (MΦs) in Th17-cell responses is still poorly understood. While neutrophils are thought to be the predominant effector of Th17-cell responses, IL-17 is also known to induce myelotropic chemokines and growth factors. Other T-cell-derived cytokines induce non-classical functions, suggesting that IL-17 sigxnaling may similarly elicit unique MΦ functions. Here, we characterized the expression of subunits of the IL-17 receptor on primary murine MΦs from different anatomical compartments. The greatest expression of IL-17 receptors was observed on mucosal Ly6C(hi) "inflammatory" MΦs. We further observed upregulation of IL-17 receptors in vitro on bone marrow-derived macrophages (BMMΦs) in response to peptidoglycan or CpG oligonucleotide stimuli, and in vivo, upon CFA administration. Macrophages expressing IL-17 receptors were observed infiltrating the hearts of mice with myocarditis, and genetic ablation of IL-17RA altered MΦ recruitment. Treating primary MΦs from a wide variety of different anatomic sources (as well as cell lines) with IL-17A induced the production of unique profiles of cytokines and chemokines, including GM-CSF, IL-3, IL-9, CCL4/MIP-1β and CCL5/RANTES. IL-17A also induced production of IL-12p70; IL-17-signaling-deficient MΦs elicited diminished IFN-γ production by responding DO11.10 CD4(+) T cells when used as APCs. These data indicate that MΦs from different anatomic locations direct IL-17-mediated responses.

The ability of CD8+ T cells to kill intracellular pathogens depends upon their capacity to attract infected cells as well as their secretion of cytolytic and antimicrobial effector molecules. We examined the Ag-induced expression of three immune effector molecules contained within cytoplasmic granules of human CD8+ T cells: the chemokine CCL5, the cytolytic molecule perforin, and the antimicrobial protein granulysin. Macrophages infected with virulent Mycobacterium tuberculosis triggered the expression of CCL5 in CD8+ T cells only in donors with previous exposure to the tuberculosis bacteria, not in naive donors. Functionally, CCL5 efficiently attracted M. tuberculosis-infected macrophages, but failed to exert direct antibacterial activity. Infected macrophages also triggered the expression of granulysin in CD8+ T cells, and granulysin was found to be highly active against drug-susceptible and drug-resistant M. tuberculosis clinical isolates. The vast majority of CCL5-positive cells coexpressed granulysin and perforin. Taken together, this report provides evidence that a subset of CD8+ T cells coordinately expresses CCL5, perforin and granulysin, thereby providing a host mechanism to attract M. tuberculosis-infected macrophages and kill the intracellular pathogen.