Regulated On Activation Normal T Cell Expressed And Secreted Research Articles

Loss of intraepidermal nerve fiber density during SIV peripheral neuropathy is mediated by monocyte activation and elevated monocyte chemotactic proteins.

BackgroundPeripheral neuropathy (PN) continues to be a major complication of human immunodeficiency virus (HIV) infection despite successful anti-retroviral therapy. Human HIV-PN can be recapitulated in a CD8-depleted, simian immunodeficiency virus (SIV)-infected rhesus macaque animal model, characterized by a loss of intraepidermal nerve fiber density (IENFD) and damage to the dorsal root ganglia (DRG). Increased monocyte traffic to the DRG has previously been associated with severe DRG pathology, as well as a loss in IENFD. Here, we sought to characterize the molecular signals associated with monocyte activation and trafficking to the DRGs.MethodsEleven SIV-infected CD8-depleted rhesus macaques were compared to four uninfected control animals. sCD14, sCD163, sCD137, regulated on activation normal T cell expressed and secreted (RANTES), and monocyte chemoattractant protein 1 (MCP-1) were measured in plasma and the latter three proteins were also quantified in DRG tissue lysates. All SIV-infected animals received serial skin biopsies to measure IENFD loss as well as BrdU inoculations to measure monocyte turnover during the course of infection. The number of BrdU+ and CD14+ CD16+ peripheral blood monocytes was determined by flow cytometry. The number of MAC387+, CCR2+, CCR5+, and CD137+ cells in DRG tissue was quantified by immunohistochemistry.ResultssCD14, sCD163, MCP-1, and sCD137 increased significantly in plasma from pre-infection to necropsy. Plasma sCD163 and RANTES inversely correlated with IENFD. Additionally, sCD137 in DRG tissue lysate was elevated with severe DRG pathology and associated with the recruitment of MAC387+ cells to DRG. Elevated numbers of CCR5+ and CCR2+ satellite cells in the DRG were found, suggesting a chemotactic role of their ligands, RANTES, and MCP-1 in recruiting monocytes to the tissue.ConclusionsWe characterized the role of systemic (plasma) and tissue-specific (DRG) monocyte activation and associated cytokines in the pathogenesis of SIV-PN. We identified sCD163 and RANTES as potential biomarkers for HIV-PN, as these were associated with a loss of IENFD. Additionally, we identified CD137 signaling to play a role in MAC387+ cell traffic to DRG and possibly contribute to severe pathology. These studies highlight the role of monocyte activation and traffic in the pathogenesis of SIV-PN, while identifying specific signaling proteins for future pharmacological blockade.

Cytokine and Chemokine Profile in Amicrobial Pustulosis of the Folds

Autoinflammation has recently been suggested in the pathogenesis of neutrophilic dermatoses but systematic studies on their cytokine profile are lacking. Notably, amicrobial pustulosis of the folds (APF), classified among neutrophilic dermatoses, has been studied only in small case series.In our University Hospital, we conducted an observational study on 15 APF patients, analyzing their clinical and laboratory features with a follow-up of 9 months to 20 years. Skin cytokine pattern of 9 of them was compared to that of 6 normal controls.In all patients, primary lesions were pustules symmetrically involving the skin folds and anogenital region with a chronic-relapsing course and responding to corticosteroids. Dapsone, cyclosporine, and tumor necrosis factor blockers were effective in refractory cases. In skin samples, the expressions of interleukin (IL)-1β, pivotal cytokine in autoinflammation, and its receptors I and II were significantly higher in APF (P = 0.005, 0.018, and 0.034, respectively) than in controls. Chemokines responsible for neutrophil recruitment such as IL-8 (P = 0.003), CXCL 1/2/3 (C-X-C motif ligand 1/2/3) (P = 0.010), CXCL 16 (P = 0.045), and RANTES (regulated on activation, normal T cell expressed and secreted) (P = 0.034) were overexpressed. Molecules involved in tissue damage like matrix metalloproteinase-2 (MMP-2) (P = 0.010) and MMP-9 (P = 0.003) were increased.APF is a pustular neutrophilic dermatosis with a typical distribution in all patients. The disorder may coexist with an underlying autoimmune/dysimmune disease but is often associated only with a few autoantibodies without a clear autoimmunity. The overexpression of cytokines/chemokines and molecules amplifying the inflammatory network supports the view that APF has an important autoinflammatory component.

The novel anti-adipogenic effect and mechanisms of action of SGI-1776, a Pim-specific inhibitor, in 3T3-L1 adipocytes.

The proviral integration site for moloney murine leukemia virus (Pim) kinases, consisting of Pim-1, Pim-2 and Pim-3, belongs to a family of serine/threonine kinases that are involved in controlling cell growth and differentiation. Pim kinases are emerging as important mediators of adipocyte differentiation. SGI-1776, an inhibitor of Pim kinases, is widely used to assess the physiological roles of Pim kinases, particularly cell functions. In the present study, we examined the effects of SGI-1776 on adipogenesis. The anti‑adipogenic effect of SGI‑1776 was measured by Oil Red O staining and AdipoRed assays. The effect of SGI‑1776 on the growth of 3T3‑L1 adipocytes was determined by cell count analysis. The effects of SGI‑1776 on the protein and mRNA expression of adipogenesis-related proteins and adipokines in 3T3‑L1 adipocytes were also evaluated by western blot analysis and RT‑PCR, respectively. Notably, SGI-1776 markedly inhibited lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. On a mechanistic level, SGI-1776 inhibited not only the expression of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ) and fatty acid synthase (FAS), but also the phosphorylation of signal transducer and activator of transcription-3 (STAT-3). Moreover, SGI-1776 decreased the expression of adipokines, including the expression of leptin and regulated on activation, normal T cell expressed and secreted (RANTES) during adipocyte differentiation. These findings demonstrate that SGI-1776 inhibits adipogenesis by downregulating the expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS and STAT-3.

Chemokine profiles of human visceral adipocytes from cryopreserved preadipocytes: Neutrophil activation and induction of nuclear factor-kappa B repressing factor.

In obesity, infiltration of adipose tissue by proinflammatory immune cells causes chronic low-grade inflammation. We investigated the chemokine profiles of human visceral adipocytes by the reverse transcription polymerase chain reaction and the effect of human neutrophil elastase (HNE) on monocyte chemoattractant protein-1 (MCP-1) mRNA and protein levels. Human adipocytes were obtained from cryopreserved omental preadipocytes of subjects with a body mass index (BMI) <30kg/m(2) or >30kg/m(2) and were cultured to assess chemokine production. Chemokine responses associated with obesity-related inflammation were well preserved in cultured human adipocytes derived from cryopreserved preadipocytes. Visceral adipocytes from subjects with a BMI >30kg/m(2) expressed mRNA for MCP-1, regulated on activation, normal T cell expressed and secreted (RANTES), epithelial cell-derived neutrophil-activating peptide-78 (ENA-78), interleukin-8 (IL-8), lymphotactin-β, and fractalkine. Although visceral adipocytes from subjects with a BMI <30kg/m(2) also expressed MCP-1, RANTES, ENA-78, and IL-8 mRNA, neither lymphotactin-β nor fraktalkine mRNA was detected. Interestingly, expression of MCP-1 mRNA was decreased significantly after exposure to HNE (85×10(3)μM/L), suggesting the induction of nuclear factor-kappa B repressing factor. Adipocytes from subjects with a BMI >30kg/m(2) or <30kg/m(2) have different chemokine profiles. Only adipocytes from subjects with a BMI >30kg/m(2) express lymphotactin-β and fractalkine mRNA. Differential chemokine profiles of visceral adipocytes contribute to infiltration of adipose tissue by adaptive immune cells. Neutrophil activation is involved in induction of nuclear factor-kappa B repressing factor, resulting in regulation of immune cell trafficking.

Ethanol extracts of Sanguisorba officinalis L. suppress TNF-α/IFN-γ-induced pro-inflammatory chemokine production in HaCaT cells

BackgroundSanguisorba officinalis L. (SOL) is a perennial plant widely distributed in Asia, its roots are well-known as a traditional herbal medicine to treat burns, chronic intestinal infections, scalds, and inflammation in Korea. Also, the roots of SOL are used for treatment of many types of allergic skin diseases, including urticarial, eczema, and allergic dermatitis. PurposeIn this study we investigated the underlying mechanism of anti-inflammatory effect of an ethanol extract of SOL roots (ESOL). Study designThe ability of ESOL to inhibit inflammatory skin disorder was tested in human keratinocyte HaCaT cells. MethodsViability test using MTT assay were used to determine non-cytotoxic concentrations of ESOL on HaCaT cells. ESOL-mediated inhibition of the tumor necrosis factor (TNF)-α/interferon (IFN)-γ-induced production of pro-inflammatory chemokines—such as macrophage-derived chemokine (MDC), regulated on activation, normal T-cell expressed and secreted (RANTES), interleukin (IL)-8, and thymus and activation regulated chemokine (TARC)—at the mRNA level was determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). The ability of ESOL to reduce the expression of pro-inflammatory marker proteins was evaluated by Western blot analysis and immunocytochemistry. ResultsESOL reduced the production of MDC, RANTES, IL-8, and TARC in HaCaT cells stimulated with TNF-α/IFN-γ at both protein and mRNA levels. ESOL also suppressed the phosphorylation of signal transducer and activator of transcription (STAT)-1, extracellular signal-regulated kinase (ERK), and inhibited both nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha (IκB-α) degradation and the nuclear translocation of NF-κB/p65. ESOL exerts anti-inflammatory effects by suppressing the expression of TNF-α/IFN-γ-stimulated chemokines and pro-inflammatory molecules via a blockade NF-κB, STAT-1, and ERK activation. ConclusionOur results suggest the preventive potential of ESOL as a herbal medicine for the treatment of inflammatory skin diseases.

Multitargeting of selected prostanoid receptors provides agents with enhanced anti-inflammatory activity in macrophages.

A polypharmacologic approach to prostanoid based anti-inflammatory therapeutics was undertaken in order to exploit both the anti- and proinflammatory properties attributed to the various prostanoid receptors. Multitargeting of selected prostanoid receptors yielded a prototype compound, compound 1 (AGN 211377), that antagonizes prostaglandin D2 receptors (DPs) DP1 (49) and DP2 (558), prostaglandin E2 receptors (EPs) EP1 (266) and EP4 (117), prostaglandin F2α receptor (FP) (61), and thromboxane A2 receptor (TP) (11) while sparing EP2, EP3, and prostaglandin I2 receptors (IPs); Kb values (in nanomoles) are given in parentheses. Compound 1 evoked a pronounced inhibition of cytokine/chemokine secretion from lipopolysaccharide or TNF-α stimulated primary human macrophages. These cytokine/chemokines included cluster of designation 40 receptor (CD40), epithelial-derived neutrophil-activating protein 78 (ENA-78), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), IL-8, IL-18, monocyte chemotactic protein-1 (CCL2) (MCP-1), tissue plasminogen activator inhibitor (PAI-1), and regulated on activation, normal T cell expressed and secreted (RANTES). In contrast, the inhibitory effects of most antagonists selective for a single receptor were modest or absent, and selective EP2 receptor blockade increased cytokine release in some instances. Compound 1 also showed clear superiority to the cyclooxygenase inhibitors diclofenac and rofecoxib. These findings reveal that blockade of multiple prostanoid receptors, with absent antagonism of EP2 and IP, may provide more effective anti-inflammatory activity than global suppression of prostanoid synthesis or highly selective prostanoid receptor blockade. These investigations demonstrate the first working example of prostanoid receptor polypharmacology for potentially safer and more effective anti-inflammatory therapeutics by blocking multiple proinflammatory receptors while sparing those with anti-inflammatory activity.

Expression of adhesion molecules, chemokines and matrix metallo- proteinases (MMPs) in viable and degenerating stage of Taenia solium metacestode in swine neurocysticercosis

Neurocysticercosis (NCC) is a parasitic infection of central nervous system (CNS). Expression of adhesion molecules, chemokines and matrix metalloproteinases (MMPs) were investigated on brain tissues surrounding viable (n=15) and degenerating cysticerci (n=15) of Taenia solium in swine by real-time RT-PCR and ELISA. Gelatin gel zymography was performed for MMPs activity. ICAM-1 (intercellular adhesion molecule-1), E-selectin, MIP-1α (macrophage inflammatory protein-1α), Eotaxin-1 and RANTES (regulated on activation, normal T cell expressed and secreted) were associated with degenerating cysticerci (cysts). However, VCAM-1 (vascular cell adhesion molecule-1), MCP-1 (monocyte chemotactic protein-1), MMP-2 and MMP-9 were associated with both viable and degenerating cysts. In conclusion, viable and degenerating cysticerci have different immune molecule profiles and role of these molecules in disease pathogenesis needs to be investigated.

Enterovirus 71 infection caused neuronal cell death and cytokine expression in cultured rat neural cells.

Fatal enterovirus type-71 (EV71) cases are associated with central nervous system infection characterized by inflammatory cell infiltration and activation, cytokine overproduction, and neuronal cell death. Although EV71 antigen has been detected in neurons and glia, the molecular mechanisms underlying EV71-associated neuroinflammation and neuronal cell death are not fully understood. Using cultured rodent neural cell models, we found that EV71 infection preferentially caused cell death in neurons but not brain-resident immune cells astrocytes and microglia. Neurons, astrocytes, and microglia responded to EV71 infection by releasing distinct profiles of cytokines, including nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, regulated on activation normal T cell expressed and secreted (RANTES), and glutamate. EV71 infection-induced neuronal cell death correlated well with the elevated production of NO, TNF-α, IL-1β, and glutamate as well as activation of microglia. Exogenous addition studies further demonstrated the neurotoxic potential of NO, TNF-α, IL-1β, and glutamate. EV71 infection-induced cytokine expression was accompanied by activation of protein tyrosine phosphorylation, mitogen-activated protein kinases (MAPKs), and NF-κB. Intriguingly, EV71 susceptibility was accompanied by infection-elevated neuronal human scavenger receptor class B member 2 expression in cultured neural cells with age-dependent manner. Biochemical and pharmacological studies revealed that after EV71 infection, microglia and accompanied cytokines play an active role in triggering bystander damage to neurons involving the tyrosine kinase/MAPKs/NF-κB signaling cascade. These data suggest that bystander damage caused by activated glia particularly the microglia could be an alternative mechanism of EV71-associated neuronal cell death. However, its clinical importance and implication require further investigation.

Cytokines in Machado Joseph Disease/Spinocerebellar Ataxia 3.

The aim of the present study is to describe the serum concentrations of a broad spectrum of cytokines in symptomatic and asymptomatic carriers of Machado Joseph disease (SCA3/MJD) CAG expansions. Molecularly confirmed carriers and controls were studied. Age at onset, disease duration, and clinical scales Scale for the Assessment and Rating of Ataxia (SARA), Neurological Examination Score for Spinocerebellar Ataxias (NESSCA), SCA Functional Index (SCAFI), and Composite Cerebellar Functional Score (CCFS) were obtained from the symptomatic carriers. Serum was obtained from all individuals and a cytokine panel "consisted of" eotaxin, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-α, IFN-γ, interleukin (IL)-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, interferon gamma-induced protein (IP)-10, monocyte chemoattractantprotein (MCP)-1, monokine induced by gamma interferon (MIG), macrophage inflammatory protein (MIP)-a, MIP-b,regulated on activation, normal T cell expressed and secreted (RANTES) and tumor necrosis factor (TNF)-α was analyzed. In a subgroup of symptomatic carriers, the cytokine panel was repeated after 360days. Cytokine distribution among groups was studied by discriminant analysis; changes in serum levels after 360days were studied by generalized estimation equation. Sixty-six symptomatic carriers, 13 asymptomatic carriers, and 43 controls were studied. No differences in cytokine patterns were found between controls and carriers of the CAG expansions or between controls and symptomatic carriers only. In contrast, eotaxin concentrations were significantly higher in asymptomatic than in symptomatic carriers or in controls (p = 0.001, ANCOVA). Eotaxin did not correlate with age, disease duration, CAG expansion, NESSCA score, and SARA score. Among symptomatic carriers, eotaxin dropped after 360days (p = 0.039, GEE). SCA3/MJD patients presented a benign pattern of serum cytokines. In contrast, levels of eotaxin, a peptide secreted by astrocytes, were elevated in the asymptomatic carriers, suggesting that a specific response of these cells can be related to symptom progression, in SCA3/MJD.

RANTES and SDF-1 Are Keys in Cell-based Therapy of TMJ Osteoarthritis

The present study aimed to investigate the therapeutic effect of injections of local bone marrow mesenchymal stem cells (BMSCs) on osteoarthritis (OA) of the temporomandibular joint (TMJ) and to explore the role of stromal cell–derived factor 1 (SDF-1) and regulated on activation, normal T-cell expressed and secreted (RANTES) in this effect. Fundamentally, OA of the TMJ was induced by unilateral anterior crossbite in mice. Exogenous green fluorescent protein–labeled BMSCs (GFP-BMSCs) were weekly injected into the TMJ region for 4, 8, and 12 wk. The reparative effects of exogenous GFP-BMSCs were investigated by morphological observation and micro–computed tomography. The differentiation of GFP-BMSCs in the cartilage was examined by double immunofluorescence of GFPs with type II collagen, and the expression of related factors in the condylar cartilage was quantified by real-time polymerase chain reaction. The role of RANTES and SDF-1 in the therapeutic effect of exogenous BMSCs was examined by both in vitro and in vivo studies. The OA cartilage of the TMJ displays a synchronous increase in SDF-1 and RANTES expression and a higher capability of attracting the migration of GFP-BMSCs. The implanted GFP-BMSCs differentiated into type II collagen–positive cells and reversed cartilage degradation and subchondral bone loss in mice with OA of the TMJ. The migration of GFP-BMSCs towards OA cartilage and the rescuing effect of GFP-BMSC injections were impaired by the inhibitors of C-X-C chemokine receptor type 4 (CXCR4) and C-C chemokine receptor type 1 (CCR1), which are the receptors of SDF-1 and RANTES, respectively. Our data indicated that SDF-1/CXCR4 and RANTES/CCR1 signals are pivotal and function synergistically in the recruitment of GFP-BMSCs towards degraded cartilage in mice OA of the TMJ.

Elevated Urinary T Helper 1 Chemokine Levels in Newly Diagnosed Hypertensive Obese Children

Objective:Increasing evidence suggests that T helper (Th) cells play a significant role in the pathogenesis of hypertension. The aim of this study was to evaluate the effect of obesity and anti-hypertensive treatment on urinary Th1 chemokines.Methods:The study groups consisted of three types of patients: hypertensive obese, healthy, and non-hypertensive obese. Pre-treatment and post-treatment samples of the hypertensive obese group and one sample from the other two groups were evaluated for urinary chemokine: regulated on activation, normal T cell expressed and secreted (RANTES), interferon-gamma-inducible protein 10 (IP10), and monokine induced by interferon-gamma (MIG). In the hypertensive obese group, urine microalbumin: creatinine ratio was examined before and after treatment. We recommended lifestyle changes to all patients. Captopril was started in those who could not be controlled with lifestyle changes and those who had stage 2 hypertension.Results:Twenty-four hypertensive obese (mean age 13.1), 27 healthy (mean age 11.2) and 22 non-hypertensive obese (mean age 11.5) children were investigated. The pre-treatment urine albumin: creatinine ratio was positively correlated with pre-treatment MIG levels (r=0.41, p<0.05). RANTES was significantly higher in the pre-treatment hypertensive and non-hypertensive obese group than in the controls. The urinary IP10 and MIG levels were higher in the pre-treatment hypertensive obese group than in the non-hypertensive obese. Comparison of the pre- and post-treatment values indicated significant decreases in RANTES, IP10, and MIG levels in the hypertensive obese group (p<0.05).Conclusion:Th1 cells could be activated in obese hypertensive children before the onset of clinical indicators of target organ damage. Urinary RANTES seemed to be affected by both hypertension and obesity, and urinary IP10 and MIG seemed to be affected predominantly by hypertension.

Yam storage protein dioscorins modulate cytokine gene expression in BALB/c and C57BL/6 lymphocytes

This study aimed to evaluate the effects of dioscorins isolated from 2 yam species, Dioscorea alata (Da-dioscorins) and Dioscorea japonica (Dj-dioscorins), on mouse immune activities. Results from cytokine array indicated that Dj-dioscorins increased the production of tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-3, IL-6, IL-10, IL-12p40, IL-13, monocyte chemoattractant protein (MCP)-1, regulated on activation, normal T cell expressed and secreted (RANTES), and granulocyte colony-stimulating factor (GCSF) in the spleen cells of BALB/c mice. In RAW264.7 macrophage, Da-dioscorins upregulated the expression of TNF-α, IL-1β, IL-6, IL-10, RANTES, MCP-1, and GCSF genes to a greater extent than Dj-dioscorins did. TNF-α, IL-1β, IL-6, IL-10, and RANTES gene expression was higher in spleen cells than in bone marrow and thymus cells in Da-dioscorin- or Dj-dioscorin-treated BALB/c and C57BL/6. Overall, our results suggest that dioscorins exert distinct immunomodulatory effects on mouse immune cells, and that different mouse strains respond differently to dioscorins.

Serum Inflammatory Cytokine Markers of Invasive Fungal Infection in Previously Immunocompetent Battle Casualties.

Invasive fungal infection (IFI) is described increasingly in individuals experiencing high-energy military trauma. Hallmarks of successful treatment involve aggressive surgical debridement and early initiation of systemic antimicrobial therapy. Currently, intravenous anti-fungal therapy commences based on appearance of wounds and patient's clinical course. Whereas some clinical protocols exist to predict which critically injured patients should receive anti-fungal therapies, there are no established serum markers associated with IFI. Our hypothesis is that serum inflammatory cytokines exist that can assist in identifying individuals at risk for IFI. This is a retrospective case control study at a single institution. Nine patients with IFI (Saksenaea vasiformis, Fusarium sp., Graphium sp., Scedosporium sp., Aspergillus sp., Mucor sp., and Alternaria sp.) after battlefield trauma were matched to nine individuals with similar injury patterns whose laboratory results were negative for IFI. The combination of serum inflammatory cytokines from the first and second debridements was examined with multiplex platform proteomic analysis. We defined statistical significance as a two-tailed α<0.05 after adjusting for multiple comparisons using the false discovery rate method. This model was refined further with correlation-based filter selection and the area under the curve of the receiver operating characteristics (AUROC) was tested. Both groups had similar Injury Severity Scores (ISS) (mean±standard deviation [SD]) (26.8±15.5 vs. 29.2±16.8, p=0.766). Elevated RANTES (regulated on activation, normal T cell expressed and secreted) alone (10,492.8±4,450.1 vs. 5,333.3±4,162.2, p=0.006) correlated with IFI. Also, the combination of persistent elevations in RANTES, interleukin (IL)-2R, and IL-15 was a robust model for predicting IFI with the AUROC being 0.9. Elevation in serum cytokines, particularly RANTES, correlated with IFI in this small group of patients. This demonstrates the potential of future rapid serum testing for early initiation and guidance of anti-fungal therapies.

CX3CR1 is an important surface molecule for respiratory syncytial virus infection in human airway epithelial cells.

Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children, and causes disease throughout life. Understanding the biology of infection, including virus binding to the cell surface, should help develop antiviral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin-binding domains. The G protein also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G protein binding to CX3CR1 is not important for infection of immortalized cell lines, but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines and HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was increased markedly by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and ∼50 % of RSV-infected cells in HAECs were CX3CR1+. HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES (regulated on activation normal T-cell expressed and secreted protein), IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1. Thus, the RSV G protein/CX3CR1 interaction is likely important in infection and infection-induced responses of the airway epithelium, the primary site of human infection.

A short-term mouse model that reproduces the immunopathological features of rhinovirus-induced exacerbation of COPD.

Viral exacerbations of chronic obstructive pulmonary disease (COPD), commonly caused by rhinovirus (RV) infections, are poorly controlled by current therapies. This is due to a lack of understanding of the underlying immunopathological mechanisms. Human studies have identified a number of key immune responses that are associated with RV-induced exacerbations including neutrophilic inflammation, expression of inflammatory cytokines and deficiencies in innate anti-viral interferon. Animal models of COPD exacerbation are required to determine the contribution of these responses to disease pathogenesis. We aimed to develop a short-term mouse model that reproduced the hallmark features of RV-induced exacerbation of COPD. Evaluation of complex protocols involving multiple dose elastase and lipopolysaccharide (LPS) administration combined with RV1B infection showed suppression rather than enhancement of inflammatory parameters compared with control mice infected with RV1B alone. Therefore, these approaches did not accurately model the enhanced inflammation associated with RV infection in patients with COPD compared with healthy subjects. In contrast, a single elastase treatment followed by RV infection led to heightened airway neutrophilic and lymphocytic inflammation, increased expression of tumour necrosis factor (TNF)-α, C-X-C motif chemokine 10 (CXCL10)/IP-10 (interferon γ-induced protein 10) and CCL5 [chemokine (C-C motif) ligand 5]/RANTES (regulated on activation, normal T-cell expressed and secreted), mucus hypersecretion and preliminary evidence for increased airway hyper-responsiveness compared with mice treated with elastase or RV infection alone. In summary, we have developed a new mouse model of RV-induced COPD exacerbation that mimics many of the inflammatory features of human disease. This model, in conjunction with human models of disease, will provide an essential tool for studying disease mechanisms and allow testing of novel therapies with potential to be translated into clinical practice.

Chemokine RANTES/CCL5 as an unknown link between wound healing in the jawbone and systemic disease: is prediction and tailored treatments in the horizon?

BackgroundThis research elucidates the question of whether common and widespread dental procedures (DP) like root filling (RF) and the removal of wisdom teeth (WT) contribute to chronic inflammation in the jawbone. Dentists, in carrying out these DP, can set off defective wound healing in the jawbone in ignorance of its connection to inflammatory mediators and the possibility of it being a hidden cause of chronic systemic diseases (SYD).Materials and methodsWe examined samples of the jawbone for seven cytokines by multiplex analysis in three groups of jawbone areas. In order to clarify systemic interrelations, specimens from 16 patients were analyzed in areas of former surgery in the retromolar wisdom tooth area; specimens from 16 patients were analyzed in the jawbone, apically of teeth with RF; and specimens from 19 patients were of the healthy jawbone. Each of the retromolar and the apical jawbone samples showed clinically fatty degenerated and osteonecrotic medullary changes.ResultsAll fatty necrotic and osteolytic jawbone (FDOJ) samples showed regulated on activation, normal T-cell expressed and secreted (RANTES) and fibroblast growth factor (FGF)-2 as the only extremely overexpressed cytokines. FDOJ cohorts showed a 30-fold mean overexpression of RANTES and a 20-fold overexpressed level of FGF-2 when compared to healthy controls.ConclusionsAs RANTES is discussed in the literature as a possible contributor to inflammatory diseases, and though it might have oncogenic effects, we hypothesize that FDOJ in areas of improper and incomplete wound healing in the jawbone might act as hyperactivated signaling pathways, while serving as an unknown source of “silent inflammation”. Because of the wide range of RANTES in immune diseases, treating FDOJ can cover many potential prediction or prognosis of individual outcomes.

Alantolactone from Saussurea lappa Exerts Antiinflammatory Effects by Inhibiting Chemokine Production and STAT1 Phosphorylation in TNF-α and IFN-γ-induced in HaCaT cells.

Skin inflammation is the most common condition seen in dermatology practice and can be caused by various allergic reactions and certain toxins or chemicals. In the present study, we investigated the antiinflammatory effects of Saussurea lappa, a medicinal herb, and its marker compounds alantolactone, caryophyllene, costic acid, costunolide, and dehydrocostuslactone in the HaCaT human keratinocyte cell line. HaCaT cells were stimulated with tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), and treated with S. lappa or each of five marker compounds. Chemokine production and expression were analyzed by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. Phosphorylation of signal transducer and activator of transcription (STAT) 1 was determined by immunoblotting. Stimulation with TNF-α and IFN-γ significantly increased the production of the following chemokines: thymus-regulated and activation-regulated chemokine (TARC): regulated on activation, normal T-cell expressed and secreted (RANTES): macrophage-derived chemokine (MDC): and interleukin-8 (IL-8). By contrast, S. lappa and the five marker compounds significantly reduced the production of these chemokines by TNF-α and IFN-γ-treated cells. S. lappa and alantolactone suppressed the TNF-α and IFN-γ-stimulated increase in the phosphorylation of STAT1. Our results demonstrate that alantolactone from S. lappa suppresses TNF-α and IFN-γ-induced production of RANTES and IL-8 by blocking STAT1 phosphorylation in HaCaT cells.

Specificity for a CCR5 Inhibitor Is Conferred by a Single Amino Acid Residue: ROLE OF ILE198

The chemokine receptors CCR5 and CCR2b share 89% amino acid homology. CCR5 is a co-receptor for HIV and CCR5 antagonists have been investigated as inhibitors of HIV infection. We describe the use of two CCR5 antagonists, Schering-C (SCH-C), which is specific for CCR5, and TAK-779, a dual inhibitor of CCR5 and CCR2b, to probe the CCR5 inhibitor binding site using CCR5/CCR2b chimeric receptors. Compound inhibition in the different chimeras was assessed by inhibition of chemokine-induced calcium flux. SCH-C inhibited RANTES (regulated on activation, normal T cell expressed and secreted) (CCL5)-mediated calcium flux on CCR5 with an IC50 of 22.8 nM but was inactive against monocyte chemoattractant protein-1 (CCL2)-mediated calcium flux on CCR2b. However, SCH-C inhibited CCL2-induced calcium flux against a CCR5/CCR2b chimera consisting of transmembrane domains IV-VI of CCR5 with an IC50 of 55 nM. A sequence comparison of CCR5 and CCR2b identified a divergent amino acid sequence located at the junction of transmembrane domain V and second extracellular loop. Transfer of the CCR5 sequence KNFQTLKIV into CCR2b conferred SCH-C inhibition (IC50 of 122 nM) into the predominantly CCR2b chimera. Furthermore, a single substitution, R206I, conferred partial but significant inhibition (IC50 of 1023 nM) by SCH-C. These results show that a limited amino acid sequence is responsible for SCH-C specificity to CCR5, and we propose a model showing the interaction with CCR5 Ile(198).

Temporal phenotypic features distinguish polarized macrophages in vitro

Macrophages are important in vascular inflammation and environmental factors influence macrophage plasticity. Macrophage transitions into pro-inflammatory (M1) or anti-inflammatory (M2) states have been defined predominately by measuring cytokines in culture media (CM). However, temporal relationships between cellular and secreted cytokines have not been established. We measured phenotypic markers and cytokines in cellular and CM of murine bone marrow-derived macrophages at multiple time points following stimulation with IFN-γ + LPS (M1), IL-4 (M2a) or IL-10 (M2c). Cytokines/proteins in M1-polarized macrophages exhibited two distinct temporal patterns; an early (0.5–3 h), transient increase in cellular cytokines (GM-CSF, KC-GRO, MIP-2, IP-10 and MIP-1β) and a delayed (3–6 h) response that was more sustained [IL-3, regulated on activation normal T cell expressed and secreted (RANTES), and tissue inhibitor of metalloproteinases 1 (TIMP-1)]. M2a-related cytokine/cell markers (IGF-1, Fizz1 and Ym1) were progressively (3–24 h) increased post-stimulation. In addition, novel patterns were observed. First, and unexpectedly, cellular pro-inflammatory chemokines, MCP-1 and MCP-3 but not MCP-5, were comparably increased in M1 and M2a macrophages. Second, Vegfr1 mRNA was decreased in M1 and increased in M2a macrophages. Finally, VEGF-A was increased in the CM of M1 cultures and strikingly reduced in M2a coinciding with increased Vegfr1 expression, suggesting decreased VEGF-A in M2a CM was secondary to increased soluble VEGFR1. In conclusion, macrophage cytokine production and marker expression were temporally regulated and relative levels compared across polarizing conditions were highly dependent upon the timing and location (cellular versus CM) of the sample collection. For most cytokines, cellular production preceded increases in the CM suggesting that cellular regulatory pathways should be studied within 6 h of stimulation. The divergent polarization-dependent expression of Vegfr1 may be essential to controlling VEGF potentially regulating angiogenesis and inflammatory cell infiltration in the vascular niche. The current study expands the repertoire of cytokines produced by polarized macrophages and provides insights into the dynamic regulation of macrophage polarization and resulting cytokines, proteins and gene expression that influence vascular inflammation.

Attenuation of microglial RANTES by NEMO-binding domain peptide inhibits the infiltration of CD8+ T cells in the nigra of hemiparkinsonian monkey

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Despite intense investigations, little is known about its pathological mediators. Here, we report the marked upregulation of RANTES (regulated on activation, normal T cell expressed and secreted) and eotaxin, chemokines that are involved in T cell trafficking, in the serum of hemiparkinsonian monkeys. Interestingly, 1-methyl-4-phenylpyridinium (MPP+), a Parkinsonian toxin, increased the expression of RANTES and eotaxin in mouse microglial cells. The presence of NF-κB binding sites in promoters of RANTES and eotaxin and down-regulation of these genes by NEMO-binding domain (NBD) peptide, selective inhibitor of induced NF-κB activation, in MPP+-stimulated microglial cells suggest that the activation of NF-κB plays an important role in the upregulation of these two chemokines. Consistently, serum enzyme-linked immuno assay (ELISA) and nigral immunohistochemistry further confirmed that these chemokines were strongly upregulated in MPTP-induced hemiparkinsonian monkeys and that treatment with NBD peptides effectively inhibited the level of these chemokines. Furthermore, the microglial upregulation of RANTES in the nigra of hemiparkinsonian monkeys could be involved in the altered adaptive immune response in the brain as we observed greater infiltration of CD8+ T cells around the perivascular niche and deep brain parenchyma of hemiparkinsonian monkeys as compared to control. The treatment of hemiparkinsonian monkeys with NBD peptides decreased the microglial expression of RANTES and attenuated the infiltration of CD8+ T cells in nigra. These results indicate the possible involvement of chemokine-dependent adaptive immune response in Parkinsonism.