OBJECTIVE

To assess whether serum levels of CC and CXC chemokines correlate with disease activity in patients with rheumatoid arthritis (RA), and to determine whether these effects predict clinical response.

METHODS

Serum levels of the chemokines CC (CCL2, CCL5) and CXC (CXCL8, CXCL9, CXCL10) were quantified at baseline and after 12 weeks of treatment with disease-modifying antirheumatic drugs or biologic agents in 28 patients using flow cytometry. Serum from 40 healthy individuals was collected for comparison at baseline. Response to treatment was classified according to the European League Against Rheumatism (EULAR) response criteria. Remission of disease was defined as a Disease Activity Score < 2.6.

RESULTS

The baseline serum concentrations of CC and CXC chemokines were significantly elevated in patients with active RA compared to healthy controls (p < 0.05) except for CCL2. Significant improvement in all disease activity measurements was observed after 12 weeks of treatment. Seventeen (60.7%) patients achieved good to moderate response based on the EULAR response criteria, and 5 (17.9%) patients achieved remission. The improvement in clinical activity in patients with RA was accompanied by a significant reduction in the serum concentration of CXCL9 and CXCL10 (p < 0.001). A significant reduction in the serum level of CXCL10 was also observed in the group that achieved EULAR response. Serum concentration of CCL5 remained significantly elevated in patients with RA (n = 5) who achieved remission compared to the healthy controls (p < 0.05).

CONCLUSIONS

Serum concentration of CXCL9 and CXCL10 may serve as sensitive biomarkers for disease activity in patients with RA.

Langerhans cell histiocytosis (LCH) is characterized by a clonal proliferation and retention of cells with a Langerhans cell (LC)-like phenotype at various sites within the body. The present study set out to elucidate whether aberrant expression of chemokine receptors or dysregulation of chemokine production in LCH lesions could explain abnormal retention of these cells. Immunohistochemical analysis on 13 LCH biopsies of bone, skin, and lymph node all expressed the immature dendritic cell (DC) marker CCR6 on the lesional LCs and absence of the mature DC marker CCR7. Furthermore, regardless of the tissue site, LCH lesions markedly overexpressed CCL20/MIP-3alpha, the ligand for CCR6. The lesional LCs appeared to be the source of this CCL20/MIP-3alpha production as well as other inflammatory chemokines such as CCL5/RANTES and CXCL11/I-TAC. These may explain the recruitment of eosinophils and CD4+CD45RO+ T cells commonly found in LCH lesions. The findings of this study emphasize that, despite abundant TNF-alpha, lesional LCs remain in an immature state and are induced to produce chemokines, which via autocrine and paracrine mechanisms cause not only the retention of the lesional LCs but also the recruitment and retention of other lesional cells. We postulate that the lesional LCs themselves control the persistence and progression of LCH.

It has become clear in the past years that chemokines and chemokine receptors are pivotal regulators of cellular communication and trafficking. In addition to the approximately 20 chemokine receptors that have been cloned and described, various orphan receptors with a chemokine receptor-like structure are known. We have investigated the orphan mouse chemokine receptor (L-CCR) in HEK 293 cells, a receptor that was originally described in a mouse macrophage cell line. Cells expressing this receptor show pertussis toxin-sensitive chemotaxis and small intracellular calcium transients in response to the chemokines CCL2, CCL7, CCL8, and CCL5. Biotinylated CCL2 binds to L-CCR-expressing cells, and transfection experiments with an L-CCR-green fluorescent protein fusion protein showed L-CCR expression in the membranes of recombinant HEK 293 cells. Although radioligand binding was not detected, it is suggested that L-CCR is a functional chemokine receptor.

Toll-like receptor 4 (TLR4), a component of the innate immune system, is recognized to promote tubulointerstitial inflammation in overt diabetic nephropathy (DN). However, there is no information on immune activation in resident renal cells at an early stage of human DN. In order to investigate this, we studied TLR4 gene and protein expression and TLR4 downward signaling in kidney biopsies of 12 patients with type 2 diabetes and microalbuminuria, and compared them with 11 patients with overt DN, 10 with minimal change disease (MCD), and control kidneys from 13 patients undergoing surgery for a small renal mass. Both in microalbuminuria and in overt DN, TLR4 mRNA and protein were overexpressed 4- to 10-fold in glomeruli and tubules compared with the control kidney and in MCD. In addition, NF-κB signaling was about fourfold higher in the glomeruli. TNF-α, IL6, CCR2, CCL5, and CCR5 mRNAs were markedly (about three- to fivefold) upregulated in microdissected glomeruli. While IL6, CCL2 and CCR5-mRNA, and CD68 were overexpressed in the tubulointerstitial compartment in clinical DN, they were not expressed in microalbuminuria. In a 6-year follow-up of microalbuminuric patients, glomerular TLR4 gene expression was associated with the subsequent loss of kidney function. Thus, innate immunity is activated in the glomeruli of patients with diabetic microalbuminuria. Enhanced TLR4 signaling may contribute to the progression occurring after the incipient, microalbuminuric form of nephropathy evolves to overt disease.

Most malignant cells are poorly immunogenic and fail to elicit an effective antitumor immune response. In contrast, viral infections of cells are promptly detected and eliminated by the immune system. Viral recognition critically hinges on cytosolic nucleic acid receptors that include the proinflammatory RNA helicase retinoic acid-inducible gene-I (RIG-I). Here, we show that targeted delivery of RIG-I agonists induced ovarian cancer cells to upregulate HLA class I and to secrete the proinflammatory cytokines CXCL10, CCL5, interleukin-6, tumor necrosis factor-alpha, and IFN-beta. Ovarian cancer cells stimulated via RIG-I became apoptotic and were readily phagocytosed by monocytes and monocyte-derived dendritic cells, which in turn upregulated HLA class I/II and costimulatory molecules and released CXCL10 and IFN-alpha. Our findings offer proof of principle that mimicking viral infection in ovarian cancer cells triggers an immunogenic form of tumor cell apoptosis that may enhance immunotherapy of ovarian cancer.

gamma delta T cells are a distinct subgroup of T lymphocytes that are enriched at certain anatomical localizations, such as the small intestinal epithelia and other epithelia. gamma delta T cells recognize microbial antigens, such as heat shock proteins (in mice) or phosphorylated bacterial metabolites (in humans), and control the integrity of epithelia. At the effector cell level, they share with the conventional alpha beta T lymphocytes potent cytotoxic activity and the capacity to produce a variety of cytokines, including specific cytokines such as keratinocyte growth factor. Here we summarize the current knowledge on the role of chemokines and their receptors in the migration and function of gamma delta T cells. As an example, the migration of gamma delta T cells to the small intestine is guided by the chemokine receptor CCR9 and the local expression of the corresponding ligand CCL25 (also termed thymus-expressed chemokine, TECK). Chemokine receptor expression also correlates with the functional program of T cells. In this respect, the strong expression of the MIP-1 alpha/MIP-1 beta/RANTES (CCL3/CCL4/CCL5)-receptor CCR5 correlates with a T-helper 1 phenotype of human V gamma 9V delta 2-expressing gamma delta T cells. The regulation of chemokine receptors, together with the pattern of local chemokine production, plays an important role in the localization of gamma delta T cells under physiological and pathophysiological conditions, such as infection, inflammation, and tumor defense.

The tumor-promoting chemokine CCL5 has been implicated in malignant transformation of breast epithelial cells, with studies to date focusing mainly on basal-type breast cancers. In this study, we investigated the consequences of CCL5 deletion in the MMTV-PyMT transgenic mouse model of luminal breast cancer. In this model, primary tumor burden and pulmonary metastases were reduced significantly in CCL5-deficient subjects, an effect found to be associated with a deficit of Th2 (IL4⁺CD4⁺ T) cells. Mechanistic investigations revealed that CCL5 activates CCR3, a highly expressed chemokine receptor on CD4⁺ T cells, and also boosts Gfi1 expression to promote the differentiation of Th2 cells, which enhance the prometastatic activity of tumor-associated myeloid cells. Clinically, polarization toward this immunosuppressive Th2 phenotype was also evident in patients with advanced luminal breast cancer. Thus, our findings showed that CCL5/CCR3 signaling promotes metastasis by inducing Th2 polarization of CD4⁺ T cells, with implications for prognosis and immunotherapy of luminal breast cancer.

Interleukin-10 (IL-10) modulates inflammatory responses elicited in vitro and in vivo by Borrelia burgdorferi, the Lyme disease spirochete. How IL-10 modulates these inflammatory responses still remains elusive. We hypothesize that IL-10 inhibits effector functions of multiple genes induced by B. burgdorferi in macrophages to control concomitantly elicited inflammation. Because macrophages are essential in the initiation of inflammation, we used mouse J774 macrophages and live B. burgdorferi spirochetes as the model target cell and stimulant, respectively. First, we employed transcriptome profiling to identify genes that were induced by stimulation of cells with live spirochetes and that were perturbed by addition of IL-10 to spirochete cultures. Spirochetes significantly induced upregulation of 347 genes at both the 4-h and 24-h time points. IL-10 inhibited the expression levels, respectively, of 53 and 65 of the 4-h and 24-h genes, and potentiated, respectively, at 4 h and 24 h, 65 and 50 genes. Prominent among the novel identified IL-10-inhibited genes also validated by quantitative real-time PCR (qRT-PCR) were Toll-like receptor 1 (TLR1), TLR2, IRAK3, TRAF1, IRG1, PTGS2, MMP9, IFI44, IFIT1, and CD40. Proteome analysis using a multiplex enzyme-linked immunosorbent assay (ELISA) revealed the IL-10 modulation/and or potentiation of RANTES/CCL5, macrophage inflammatory protein 2 (MIP-2)/CXCL2, IP-10/CXCL10, MIP-1α/CCL3, granulocyte colony-stimulating factor (G-CSF)/CSF3, CXCL1, CXCL5, CCL2, CCL4, IL-6, tumor necrosis factor alpha (TNF-α), IL-1α, IL-1β, gamma interferon (IFN-γ), and IL-9. Similar results were obtained using sonicated spirochetes or lipoprotein as stimulants. Our data show that IL-10 alters effectors induced by B. burgdorferi in macrophages to control concomitantly elicited inflammatory responses. Moreover, for the first time, this study provides global insight into potential mechanisms used by IL-10 to control Lyme disease inflammation.

Release of chemotactic factors in response to tissue damage has been described for different musculoskeletal tissues, including the intervertebral disc (IVD). This study investigated the chemoattractants that are released by induced degenerative IVDs and may be involved in recruiting mesenchymal stem cells (MSCs). Bovine caudal discs were cultured within a bioreactor and loaded under conditions that mimicked physiological or degenerative settings. Between days 4-6, medium was replaced by PBS, which was subsequently used for proteomic, ELISA and immunoprecipitation analyses of secreted chemokines and cytokines. A Boyden chamber assay was used to observe human MSC migration towards native and chemokine depleted media. Gene expression levels of chemokine receptors in human MSCs were analysed, and CCL5 was localised in bovine and human IVD by immunohistochemistry. Proteomic analysis revealed the presence of CCL5 and CXCL6 within conditioned media. Higher concentrations of CCL5 were found in the degenerative media, and a relationship was found between interleukin-1β and CCL5 concentration. Chemokine immunoprecipitation showed that MSCs had a significantly reduced chemotactic migration towards CCL5-immunoprecipitated and CCL5/CXCL6 co-immunoprecipitated media, whilst CXCL6 depletion did not change MSC chemotaxis. MSCs showed a significant increase in mRNA expression of the CCL5 receptors, CCR1 and CCR4, upon culture in degenerative media. Furthermore, CCL5 was identified in bovine and human disc tissue by immunohistochemistry. Hence, CCL5 may be a key chemoattractant that is produced and released by the intervertebral disc cells. Therefore, these factors could be used to enhance stem/progenitor cell mobilisation in regenerative therapies for early stages of disc degeneration.

The level of serum CCL5, a C-C chemokine, is reportedly correlated with tumor progression in several cancers. We herein investigated the mechanisms by which CCL5 might contribute to tumor progression in gastric cancer. Serum CCL5 levels significantly correlated with tumor progression and prognosis in patients with gastric cancer. Immunohistochemistry showed that tumor-infiltrating lymphocytes expressed CCL5, while the tumor cells expressed the CCL5 receptors. Fluorescent double staining showed that tumor-infiltrating CD4+ cells rather than CD8+ cells preferentially expressed CCL5. Using gastric cancer cell lines (MKN45, KATO III), we examined CCL5 production by coculturing whole peripheral blood mononuclear cells (PBMCs), CD4+ cells, or CD8+ cells, with tumor cells. CD4+ cells cocultured with tumor cells remarkably enhanced CCL5 production in a direct cell-cell contact manner over other cocultured PBMCs, including CD8+ cells. Gastric cancer cell lines expressed CCL5 receptors and augmented their proliferation in response to CCL5 stimulation. Furthermore, we examined the effect of CCL5-treated cancer cells on the cocultured PBMCs, focusing on the CD4+/CD8+ proportion and apoptosis. Coculture of CCL5-treated gastric cancer cells with PBMCs resulted in a significant decrease in the proportion of CD8+ cells but not CD4+ cells, suggesting Fas-FasL-mediated apoptosis in CD8+ cells. In immunodeficient mice coinjected with KATO III and PBMCs, neutralization of CCL5 significantly suppressed tumor progression, resulting in a favorable outcome. In conclusion, gastric cancer cells might thus induce CD4+ T cells to secrete CCL5 and exploit it for their progression, as well as to aid in the prevention of CD8+ T cell-involved tumor elimination.

Prostate cancer stem cells (PCSCs) play a critical role in prostate cancer progression and metastasis, which remains an obstacle for successful prostate cancer treatment. Tumor-associated macrophages (TAMs) are the most abundant immune cell population within the tumor microenvironment (TME). Systematic investigation of the interaction and network signaling between PCSCs and TAMs may help in searching for the critical target to suppress PCSCs and metastasis. Herein, we demonstrated that TAMs-secreted CCL5 could significantly promote the migration, invasion, epithelial-mesenchymal transition (EMT) of prostate cancer cells as well as the self-renewal of PCSCs in vitro. QPCR screening validated STAT3 as the most significant response gene in prostate cancer cells following CCL5 treatment. RNA-sequencing and mechanistic explorations further revealed that CCL5 could promote PCSCs self-renewal and prostate cancer metastasis via activating the β-catenin/STAT3 signaling. Notably, CCL5 knockdown in TAMs not only significantly suppressed prostate cancer xenografts growth and bone metastasis but also inhibited the self-renewal and tumorigenicity of PCSCs in vivo. Finally, clinical investigations and bioinformatic analysis suggested that high CCL5 expression was significantly correlated with high Gleason grade, poor prognosis, metastasis as well as increased PCSCs activity in prostate cancer patients. Taken together, TAMs/CCL5 could promote PCSCs self-renewal and prostate cancer metastasis via activating β-catenin/STAT3 signaling. This study provides a novel rationale for developing TAMs/CCL5 as a potential molecular target for PCSCs elimination and metastatic prostate cancer prevention.

BACKGROUND

Mucosal CXC chemokines recruit inflammatory cells to the infected urinary tract. The chemokine response repertoire of the urinary tract and the relationship to disease severity have not been examined, however.

METHODS

This study quantified CXC (CXCL1, CXCL3, CXCL5, CXCL8, CXCL9, and CXCL10) and CC (CCL2, CCL4, and CCL5) chemokines in sequential urine samples obtained from 50 patients with febrile urinary tract infections during 24 hours after diagnosis.

RESULTS

All patients had elevated chemokine levels, but bacteremic infections caused higher CXCL1, CXCL3, CXCL5, CXCL8, and CCL2 responses. CCL2 and CXCL8 levels were higher in patients with acute pyelonephritis symptoms and CCL2, CXCL3, CCL4, CXCL5, and CXCL10 were significantly correlated to C-reactive protein (CRP) and temperature. Women and men showed different chemokine responses.

CONCLUSIONS

Febrile urinary tract infections are accompanied by a complex chemokine response. The response magnitude reflects disease severity, and the repertoire is influenced by gender and underlying disease.

BACKGROUND

The mechanisms by which viruses induce asthma exacerbations are not well understood.

OBJECTIVE

We characterized fluctuations in nasal aspirate cytokines during naturally occurring respiratory viral infections in children with asthma.

METHODS

Sixteen children underwent home collections of nasal aspirates when they were without cold symptoms and again during self-reported respiratory illnesses. The presence of viral infection was ascertained by multiplex PCR. Cytokines were measured using multiplex immune assay. mRNA expression for selected markers of viral infection was measured using RT-PCR. A cumulative respiratory symptom score was calculated for each day of measurement. Generalized estimated equations were used to evaluate associations between viral infection and marker elevation, and between marker elevation and symptom score.

RESULTS

The 16 patients completed a total of 37 weeks of assessment (15 'well' weeks; 22 self-assessed 'sick' weeks). Viral infections were detected in 3 of the 'well' weeks and 17 of the 'sick' weeks (10 rhinovirus, three coronavirus, two influenza A, two influenza B, two respiratory syncytial virus, one parainfluenza). Compared to virus-negative well weeks, nasal aspirate IFN-γ, CXCL8/IL-8, CXCL10/IP-10, CCL5/RANTES, CCL11/eotaxin-1, CCL2/MCP-1, CCL4/MIP-1β, CCL7/MCP-3, and CCL20/MIP3α protein levels increased during virus-positive sick weeks. Only a subset of cytokines (IFN-γ, CXCL8, CCL2, CCL4, CCL5, and CCL20) correlated with self-reported respiratory tract symptoms. While many aspirates were dilute and showed no mRNA signal, viral infection significantly increased the number of samples that were positive for IFN-λ1, IFN-λ2/3, TLR3, RIG-I, and IRF7 mRNA.

CONCLUSIONS

We conclude that in children with asthma, naturally occurring viral infections apparently induce a robust innate immune response including expression of specific chemokines, IFNs, and IFN-responsive genes.

The lack of an accurate diagnosis has been a serious obstacle to the advancement of the anti-Trypanosoma cruzi chemotherapy and long-term infection can result in different health risks to human. PCRs are alternative methods, more sensitive than conventional parasitological techniques, which due to their low sensitivities are considered unsuitable for these purposes. The aim of this study was to investigate a sensitive diagnostic strategy to quantify blood and cardiac tissues parasites based on real-time PCR tools during acute and chronic phases of murine Chagas disease, as well as to monitor the evolution of infection in those mice under specific treatment. In parallel, fresh blood examination, immunological analysis and quantification of cardiac inflammation were also performed to confront and improve real-time PCR data. Similar profiles of parasitemia curves were observed in both quantification techniques during the acute phase of the infection. In contrast, parasites could be quantified only by real-time PCR at 60 and 120 days of infection. In cardiac tissue, real-time PCR detected T. cruzi DNA in 100% of infected mice, and using this tool a significant Pearson correlation between parasite load in peripheral blood and in cardiac tissue during acute and chronic phases was observed. Levels of serum CCL2, CCL5 and nitric oxide were coincident with parasite load but focal and diffuse mononuclear infiltrates was observed, even with significant (p<0.05) reduction of parasitism after 60 days of infection. Later, this methodology was used to monitor the evolution of infection in animals treated with itraconazole (Itz). Itz-treatment induced a reduction of parasite load in both blood and cardiac muscle at the treatment period, but after the end of chemotherapy an increase of parasitism was detected. Interestingly, inflammatory mediators levels and heart inflammation intensity had similar evolution to the parasite load, in the group of animals treated. Taken together, our data show that real-time PCR strategy used was suitable for studies of murine T. cruzi infection and may prove useful in investigations involving experimental chemotherapy of the disease and the benefits of treatment in relation to parasitism and inflammatory response.

Dendritic cells (DC) play a key role in the host immune response to infections. Human cytomegalovirus (HCMV) infection can inhibit the maturation of DC and impair their ability to stimulate T cell proliferation and cytotoxicity. In this study, we assessed the effects of HCMV infection on the migratory behavior of human DC. The HCMV strain TB40/E inhibited the migration of immature monocyte-derived DC in response to inflammatory chemokines by 95% 1 day after infection. This inhibition was mediated by early viral replicative events, which significantly reduced the cell-surface expression of CC chemokine receptor 1 (CCR1) and CCR5 by receptor internalization. HCMV infection also induced secretion of the inflammatory chemokines CC chemokine ligand 3 (CCL3)/macrophage inflammatory protein-1alpha (MIP-1alpha), CCL4/MIP-1beta, and CCL5/regulated on activation, normal T expressed and secreted (RANTES). Neutralizing antibodies for these chemokines reduced the effects of HCMV on chemokine receptor expression and on DC migration by approximately 60%. Interestingly, the surface expression of the lymphoid chemokine receptor CCR7 was not up-regulated after HCMV infection on immature DC, and immature-infected DC did not migrate in response to CCL19/MIP-3beta. These findings suggest that blocking the migratory ability of DC may be a potent mechanism used by HCMV to paralyze the early immune response of the host.

Staphylococcus epidermidis is a commensal on skin, whereas Staphylococcus aureus is a transient pathogen. The aim was to determine whether the skin's innate defence systems responded differently to these microorganisms. Differential gene expression of a human skin equivalent (SE) model was assessed by microarray technology, in response to colonization by S. epidermidis or S. aureus. Only a small number of transcripts were significantly (P<0.0001) increased (12) or decreased (35) with gene expression changes of >2-fold on SEs colonized with S. epidermidis compared with controls (no colonization). Expression of one innate defence gene, pentraxin 3 (PTX3), was upregulated, while psoriasin, S100A12, S100A15, beta defensin 4, beta defensin 3, lipocalin 2 and peptidoglycan recognition protein 2 were downregulated. In contrast, large numbers of transcripts were significantly increased (480) or decreased (397) with gene expression changes of >2-fold on SEs colonized with S. aureus compared with controls. There was upregulation in gene expression of many skin defence factors including Toll-like receptor 2, beta defensin 4, properdin, PTX3, proinflammatory cytokines tumour necrosis factor-alpha, IL-1 alpha, IL-1 beta, IL-17C, IL-20, IL-23A and chemokines IL-8, CCL4, CCL5, CCL20 and CCL27. These differences may partly explain why S. epidermidis is a normal skin resident and S. aureus is not.

There are a large number of stable pancreatic ductal carcinoma cell lines (PDCL) that are used by researchers worldwide. Detailed data about their differentiation status and genetic alterations are present in the literature, but a systematic correlation with cell biological behavior is often lacking. PDCL ( n=12) were clustered by source of tumor cell (ascites, primary tumor, metastasis), and the data of functional cell biology were correlated with the reported structural and genetic profiles. Major histocompatibility complex expression, chemosensitivity and aneuploidia appeared to be related to the source of PDCL, and proliferative capacity appeared to be related to the grade of differentiation. No correlation between genetic/structural features of PDCL and biological behavior was found. All the cell lines appeared generally insensitive to in vitro treatment with 5-fluorouracil and showed variable degrees of susceptibility to gemcitabine, raltitrexed and oxaliplatin. All the PDCL showed resistance to Fas-mediated apoptosis but were significantly sensitive to the pro-apoptotic effect of inflammatory cytokines [interleukin (IL)-1beta, tumor necrosis factor (TNF)alpha and interferon gamma]. PDCL were characterized for the secretion of several factors relevant to the tumor-immune cross talk. Vascular endothelial growth factor, CCL2, CCL5 and transforming growth factor beta were the factors most frequently released; less frequent was the secretion of CXCL8, CCL22, IL-6 and sporadically CXCL12, IL-10 and hepatocyte growth factor. The cytokines IL-1beta and TNFalpha were always undetectable. In conclusion, a clear correlation between structural/genetic features and function could not be detected, suggesting the weakness of a "morphological" classification for the in vitro studies of pancreatic cancer.

Posttranscriptional regulation is emerging as a key factor in glucocorticoid (GC)-mediated gene regulation. We investigated the role of the human GC receptor (GR) as an RNA-binding protein and its effect on mRNA turnover in human airway epithelial cells. Cell treatment with the potent GC budesonide accelerated the decay of CCL2 mRNA (t(1/2) = 8 ± 1 min versus 62 ± 17 min in DMSO-treated cells) and CCL7 mRNA (t(1/2) = 15 ± 4 min versus 114 ± 37 min), but not that of CCL5 mRNA (t(1/2)=231 ± 8 min versus 266 ± 5 min) in the BEAS-2B cell line. This effect was inhibited by preincubation with an anti-GR Ab, indicating that GR itself plays a role in the turnover of these transcripts. Coimmunoprecipitation and biotin pulldown experiments showed that GR associates with CCL2 and CCL7 mRNAs, but not CCL5 mRNA. These methods confirmed CCL2 mRNA targeting by GR in human primary airway epithelial cells. Association of the GR was localized to the 5' untranslated region of CCL2 mRNA and further mapped to nt 44-60. The collection of transcripts associated with GR, identified by immunoprecipitation of GR-mRNA complexes followed by microarray analysis, revealed 479 transcripts that associated with GR. Computational analysis of the primary sequence and secondary structures of these transcripts yielded a GC-rich motif, which was shown to bind to GR in vitro. This motif was used to predict binding of GR to an additional 7889 transcripts. These results indicate that cytoplasmic GR interacts with a subset of mRNA through specific sequences and can regulate turnover rates, suggesting a novel posttranscriptional role for GR as an RNA-binding protein.

OBJECTIVE

Alpha-lipoic acid (LA) is a commonly used dietary supplement that exerts anti-oxidant and anti-inflammatory effects in vivo and in vitro. We investigated the mechanisms by which LA may confer protection in models of established atherosclerosis.

METHODS

Watanabe heritable hyperlipidemic (WHHL) rabbits were fed with high cholesterol chow for 6 weeks and then randomized to receive either high cholesterol diet alone or combined with LA (20mg/kg/day) for 12 weeks. Vascular function was analyzed by myography. The effects of LA on T cell migration to chemokine gradients was assessed by Boyden chamber. NF-kappaB activation was determined by measuring translocation and electrophoresis migration shift assay (EMSA).

RESULTS

LA decreased body weight by 15+/-5% without alterations in lipid parameters. Magnetic Resonance Imaging (MRI) analysis demonstrated that LA reduced atherosclerotic plaques in the abdominal aorta, with morphological analysis revealing reduced lipid and inflammatory cell content. Consistent with its effect on atherosclerosis, LA improved vascular reactivity (decreased constriction to angiotensin II and increased relaxation to acetylcholine and insulin), inhibited NF-kappaB activation, and decreased oxidative stress and expression of key adhesion molecules in the vasculature. LA reduced T cell content in atherosclerotic plaque in conjunction with decreasing ICAM and CD62L (l-selectin) expression. These effects were confirmed by demonstration of a direct effect of LA in reducing T cell migration in response to CCL5 and SDF-1 and decreasing T cell adhesion to the endothelium by intra-vital microscopy.

CONCLUSIONS

The present findings offer a mechanistic insight into the therapeutic effects of LA on atherosclerosis.

METHODS

Laboratory study.

OBJECTIVE

To evaluate expression of chemokine regulated and normal T cell expressed and secreted (RANTES)/C-C motif ligand 5 (CCL5) and interleukins in intervertebral discs (IVDs) specimens from patients with discogram-proven painful degeneration.

BACKGROUND

Discogenic back pain results in tremendous costs related to treatment and lost productivity. The relationship between inflammation, degeneration (IVD), and cytokine upregulation is well established, but other mediators of the inflammatory cascade are not well characterized.

METHODS

Painful IVDs were taken from 18 patients undergoing surgery for discogenic pain with positive preoperative discogram. Painless control tissue was taken at autopsy from patients without back pain/spinal pathology or spinal levels with negative discograms resected for deformity.Quantitative real time polymerase chain reaction (qRT-PCR) was performed to evaluate RANTES, IL-1β, IL-6, and IL-8 expression in painful and control discs. RANTES and interleukin expression were analyzed on the basis of Pfirrmann grade.Disc cells were cultured in alginate beads using 2 groups: an untreated group and a group treated with 10 ng/mL IL-1β, 10 ng/mL TNF-α, and 1% fetal bovine serum to induce a degenerative phenotype.

RESULTS

Nine painless IVD specimens and 7 painful IVD specimens were collected. RANTES expression demonstrated a 3.60-fold increase in painful discs versus painless discs, a significant difference (P = 0.049). IL-1β expression demonstrated significantly higher expression in painful discs (P = 0.03). RANTES expression data demonstrated significant upregulation with increasing Pfirrmann grade (P = 0.045). RANTES expression correlated significantly with IL-1β expression (ρ = 0.67, P < 0.0001). RANTES expression increased more than 200-fold in the alginate culture model in cells treated with IL-1β/TNF-α, 1% fetal bovine serum (P < 0.001).

CONCLUSIONS

RANTES and IL-1β expression was significantly elevated in painful IVDs after careful selection of painless versus painful IVD tissue. RANTES expression was found to correlate significantly with expression of IL-1β. RANTES was upregulated by IL-1β/TNF-α/1% fetal bovine serum an in vitro treatment to induce a degenerative phenotype.

CCR1 has previously been shown to play important roles in leukocyte trafficking, pathogen clearance, and the type 1/type 2 cytokine balance, although very little is known about its role in the host response during sepsis. In a cecal ligation and puncture model of septic peritonitis, CCR1-deficient (CCR1(-/-)) mice were significantly protected from the lethal effects of sepsis when compared with wild-type (WT) controls. The peritoneal and systemic cytokine profile in CCR1(-/-) mice was characterized by a robust, but short-lived and regulated antibacterial response. CCR1 expression was not required for leukocyte recruitment, suggesting critical differences extant in the activation of WT and CCR1(-/-) resident or recruited peritoneal cells during sepsis. Peritoneal macrophages isolated from naive CCR1(-/-) mice clearly demonstrated enhanced cytokine/chemokine generation and antibacterial responses compared with similarly treated WT macrophages. CCR1 and CCL5 interactions markedly altered the inflammatory response in vivo and in vitro. Administration of CCL5 increased sepsis-induced lethality in WT mice, whereas neutralization of CCL5 improved survival. CCL5 acted in a CCR1-dependent manner to augment production of IFN-gamma and MIP-2 to damaging levels. These data illustrate that the interaction between CCR1 and CCL5 modulates the innate immune response during sepsis, and both represent potential targets for therapeutic intervention.

OBJECTIVE

We investigated the effect of ATP (ATP) encapsulated in liposomes (ATP-liposomes) on the level of inflammation and neuronal death in the retina induced by ischemia reperfusion (IR).

METHODS

Primary retinal ganglion cells treated with ATP-liposomes, empty liposomes, and phosphate buffer solution (PBS) were deprived of oxygen and glucose (OGD) for 6 h in vitro, in an anaerobic chamber. Plates were assessed for the proportion of necrotic versus apoptotic cells and for cell survival 12 h after OGD. For in vivo experiments, we induced retinal ischemia by unilateral elevation of intraocular pressure for 1 h by direct corneal canulation. Mice were injected with liposomes or PBS 24 h before IR, at the time of surgery, and every 24 h until sacrifice. Transmission electron microscopic analysis was used to identify necrotic and apoptotic cells in ischemic retinas. The changes in expression of pro-inflammatory genes 24 h post reperfusion were assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Corresponding changes in protein abundances were analyzed by immunohistochemistry. Cell death was evaluated by direct counting of neurons in the ganglion cell layer (GCL) of flatmounted retinas 7 days post reperfusion.

RESULTS

Treatment with ATP-liposomes increases retinal ganglion cell (RGC) survival and decreases necrotic cell death following OGD. Injection of ATP-liposomes markedly decreased necrotic cell death in the GCL following retinal ischemia. The ATP-liposome treatment reduced the expression of pro-inflammatory genes, including that of interleukin 1β (Il1β), interleukin 6 (Il6), tumor necrosis factor (Thf), chemokine (C-C motif) ligand 2 (Ccl2), chemokine (C-C motif) ligand 5 (Ccl5), chemokine (C-X-C motif) ligand 10 (Cxcl10), intercellular adhesion molecule 1 (Icam1), and nitric oxide synthase 2 (Nos2), in the retina 24 h after IR and significantly reduced the GCL neuron death rate 7 days after reperfusion.

CONCLUSIONS

ATP-liposome treatment of IR-challenged neural tissues suppressed necrosis and correlated with a significantly reduced level of inflammation and retinal damage.

Dengue virus (DENV), a mosquito-borne flavivirus, is a public health problem in many tropical countries. Recent clinical data have shown an association between levels of different chemokines in plasma and severity of dengue. We evaluated the role of CC chemokine receptors CCR1, CCR2 and CCR4 in an experimental model of DENV-2 infection in mice. Infection of mice induced evident clinical disease and tissue damage, including thrombocytopenia, hemoconcentration, lymphopenia, increased levels of transaminases and pro-inflammatory cytokines, and lethality in WT mice. Importantly, infected WT mice presented increased levels of chemokines CCL2/JE, CCL3/MIP-1α and CCL5/RANTES in spleen and liver. CCR1⁻/⁻ mice had a mild phenotype with disease presentation and lethality similar to those of WT mice. In CCR2⁻/⁻ mice, lethality, liver damage, levels of IL-6 and IFN-γ, and leukocyte activation were attenuated. However, thrombocytopenia, hemoconcentration and systemic TNF-α levels were similar to infected WT mice. Infection enhanced levels of CCL17/TARC, a CCR4 ligand. In CCR4⁻/⁻ mice, lethality, tissue injury and systemic inflammation were markedly decreased. Despite differences in disease presentation in CCR-deficient mice, there was no significant difference in viral load. In conclusion, activation of chemokine receptors has discrete roles in the pathogenesis of dengue infection. These studies suggest that the chemokine storm that follows severe primary dengue infection associates mostly to development of disease rather than protection.

Inflammatory illness is associated with depression. Preclinical work has shown that chemokines are linked with peripheral-central crosstalk and may be important in mediating depressive behaviours. We sought to establish what evidence exists that differences in blood or cerebrospinal fluid chemokine concentration discriminate between individuals with depression and those without. Following PRISMA guidelines, we systematically searched Embase, PsycINFO and Medline databases. We included participants with physical illness for subgroup analysis, and excluded participants with comorbid psychiatric diagnoses. Seventy-three studies met the inclusion criteria for the meta-analysis. Individuals with depression had higher levels of blood CXCL4 and CXCL7 and lower levels of blood CCL4. Sensitivity analysis of studies with only physically healthy participants identified higher blood levels of CCL2, CCL3, CCL11, CXCL7 and CXCL8 and lower blood levels of CCL4. All other chemokines examined did not reveal significant differences (blood CCL5, CCL7, CXCL9, CXCL10 and cerebrospinal fluid CXCL8 and CXCL10). Analysis of the clinical utility of the effect size of plasma CXCL8 in healthy individuals found a negative predictive value 93.5%, given the population prevalence of depression of 10%. Overall, our meta-analysis finds evidence linking abnormalities of blood chemokines with depression in humans. Furthermore, we have demonstrated the possibility of classifying individuals with depression based on their inflammatory biomarker profile. Future research should explore putative mechanisms underlying this association, attempt to replicate existing findings in larger populations and aim to develop new diagnostic and therapeutic strategies.