IFN-gamma secretion by natural killer (NK) cells is pivotal to several tumor and viral immune responses, during which NK and dendritic cells cooperation is required. We show here that macrophages are mandatory for NK cell IFN-gamma secretion in response to erythrocytes infected with Plasmodium falciparum (Pf), a causative agent of human malaria. In addition, direct sensing of Pf infection by NK cells induces their production of the proinflammatory chemokine CXCL8, without triggering their granule-mediated cytolytic programs. Despite their reported role in Pf recognition, Toll-like receptor (TLR) 2, TLR9, and TLR11 are individually dispensable for NK cell activation induced by Pf-infected erythrocytes. However, IL-18R expression on NK cells, IL-18 production by macrophages, and MyD88 on both cell types are essential components of this previously undescribed pathway of NK cell activation in response to a parasite infection.

Platelet factor-4 (PF-4)/CXCL4 was the first chemokine described to inhibit neovascularization. Here, the product of the nonallelic variant gene of CXCL4, PF-4var1/PF-4alt, designated CXCL4L1, was isolated for the first time from thrombin-stimulated human platelets and purified to homogeneity. Although secreted CXCL4 and CXCL4L1 differ in only three amino acids, CXCL4L1 was more potent in inhibiting chemotaxis of human microvascular endothelial cells toward interleukin-8 (IL-8)/CXCL8 or basic fibroblast growth factor (bFGF). In vivo, CXCL4L1 was also more effective than CXCL4 in inhibiting bFGF-induced angiogenesis in rat corneas. Thus, activated platelets release CXCL4L1, a potent regulator of endothelial cell biology, which affects angiogenesis and vascular diseases.

OBJECTIVE

To determine the proinflammatory cytokine profile of aqueous humor from glaucomatous eyes.

METHODS

Aqueous humor samples were prospectively collected from 38 eyes (26 primary open angle glaucoma [POAG] and 12 primary angle closure glaucoma [PACG] eyes) of 37 medically treated glaucoma patients and 23 cataract subjects recruited in an institutional setting in this case-controlled study. The main outcome measure was to quantify the levels of 29 inflammatory cytokines in the aqueous of glaucoma and cataract subjects using a multiplexed cytokine analysis. Data on patient demographics, duration of glaucoma, preoperative intraocular pressure (IOP) as well as duration of anti-glaucoma therapy were also collected for correlation analysis.

RESULTS

Mean duration of glaucoma was 53.8 months (range 1-360 months). Aqueous obtained from the glaucoma patients showed increased concentration of interleukin (IL)-9 (p=0.032), IL-12 (p=0.003), interferon (IFN)-α (p=0.034), IFN-γ (p=0.002), monokine induced by interferon-gamma (MIG or CXCL9) (p=0.006), and IL-10 (p=0.050), compared to the cataract group. The POAG group had higher IL-12 (p=0.011), IFN-γ (p=0.005), and CXCL9 (p=0.047) levels than controls, while the PACG group had higher interleukin-8 (CXCL8) (p=0.015) and CXCL9 (p=0.023) levels than the controls. No significant correlation was observed between aqueous cytokine level and preoperative IOP and duration of glaucoma. Duration of topical Timolol and Alphagan therapy correlated negatively with CXCL8 (r=-0.588, p=0.035), respectively.

CONCLUSIONS

Primary glaucoma is associated with an aqueous inflammatory response and this is different between POAG and PACG groups. Duration of glaucoma treatment may have an effect on cytokine profile in the aqueous.

BACKGROUND

The cause of corticosteroid-resistant (CR) asthma is unknown.

OBJECTIVE

We sought to perform gene microarray analyses by using bronchoalveolar lavage (BAL) cells from well-characterized subjects with CR asthma and subject with corticosteroid-sensitive (CS) asthma to elucidate the differential expression of genes that contribute to the development of corticosteroid resistance.

METHODS

The patients were characterized as having CR or CS asthma based on FEV(1) percent predicted improvement after a 1-week course of oral prednisone. Expression of selected gene targets was verified by means of real-time PCR and ELISA.

RESULTS

Microarray analyses demonstrated significantly higher levels (>3-fold increase, P < .05) of transcripts for TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, CXCL1, CXCL2, CXCL3, CXCL8 (IL-8), CCL3, CCL4, and CCL20 in BAL cells of subjects with CR asthma. These findings, confirmed by means of RT-PCR in additional BAL samples, were consistent with classical macrophage activation by bacterial products. In contrast, markers of alternatively activated macrophages, arginase I and CCL24, were decreased. Genes associated with activation of the LPS signaling pathway (early growth response 1, dual-specificity phosphatase 2, molecule possessing ankyrin repeats induced by LPS, and TNF-alpha-induced protein 3) were significantly increased in BAL samples from subjects with CR asthma (P < .05). These patients had significantly higher amounts (1444.0 +/- 457.3 pg/mg total protein) of LPS in BAL fluid than seen in subjects with CS asthma (270.5 +/- 216.0 pg, P < .05), as detected by using the LAL assay and confirmed by means of gas chromatographic/mass spectrometric analysis. Prolonged exposure to LPS induced functional steroid resistance to dexamethasone in normal human monocytes, as demonstrated by persistently increased IL-6 levels in the presence of dexamethasone.

CONCLUSIONS

Classical macrophage activation and induction of LPS signaling pathways along with high endotoxin levels detected in BAL fluid from subjects with CR asthma suggest that LPS exposure might contribute to CR asthma.

COPD is a disease of innate immunity and bacterial infections are a dominant cause of exacerbations in the later stages resulting in poor health and high mortality. The pathogen-associated molecular pattern (PAMP) lipopolysaccharide (LPS) is sensed by immune cells through activation of the toll-like receptor 4 (TLR4). This leads to the activation of NADPH oxidase (NOX) and NF-κB which together drive COPD inflammation. In this study we show in human PBMCs that LPS stimulated proinflammatory cytokine release (CXCL8 and IL6) was inhibited by approximately 50% by the broad specificity phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin. Our results also demonstrate that activation of PI3K following LPS stimulation is mediated by a NOX4 dependent mechanism releasing endogenous H2O2, as the NOX4 inhibitor apocynin blocked LPS induced AKT phosphorylation. Moreover, LPS-induced PI3K activation was inhibited by the anti-oxidant N-acetylcysteine in a concentration dependent manner (IC50 ~100 μM). In addition, our data demonstrated that inhibition of small G proteins, by pre-treatment with pertussis toxin, inhibited LPS-induced AKT phosphorylation. Furthermore, the G-protein inhibitors pertussis toxin and mastoparan both inhibited LPS-induced CXCL8 and IL-6 release by approximately 50%. Together, these data indicate there is a mechanism in human PBMCs where TLR4 activation by LPS leads to ROS generation through NOX4 and activation of the PI3K pathway. This effect is apparently mediated through small G proteins facilitating the release of pro-inflammatory cytokines.

Psoriasis is characterized by a specific microRNA expression profile, distinct from that of healthy skin. MiR-31 is one of the most highly overexpressed microRNAs in psoriasis skin; however, its biological role in the disease has not been studied. In this study, we show that miR-31 is markedly overexpressed in psoriasis keratinocytes. Specific inhibition of miR-31 suppressed NF-κB-driven promoter luciferase activity and the basal and TNF-α-induced production of IL-1β, CXCL1/growth-related oncogene-α, CXCL5/epithelial-derived neutrophil-activating peptide 78, and CXCL8/IL-8 in human primary keratinocytes. Moreover, interference with endogenous miR-31 decreased the ability of keratinocytes to activate endothelial cells and attract leukocytes. By microarray expression profiling, we identified genes regulated by miR-31 in keratinocytes. Among these genes, we identified serine/threonine kinase 40 (STK40), a negative regulator of NF-κB signaling, as a direct target for miR-31. Silencing of STK40 rescued the suppressive effect of miR-31 inhibition on cytokine/chemokine expression, indicating that miR-31 regulates cytokine/chemokine expression via targeting STK40 in keratinocytes. Finally, we demonstrated that TGF-β1, a cytokine highly expressed in psoriasis epidermis, upregulated miR-31 expression in keratinocytes in vitro and in vivo. Collectively, our findings suggest that overexpression of miR-31 contributes to skin inflammation in psoriasis lesions by regulating the production of inflammatory mediators and leukocyte chemotaxis to the skin. Our data indicate that inhibition of miR-31 may be a potential therapeutic option in psoriasis.

OBJECTIVE

To investigate the possible role of chemokines and cytokines in the pathogenesis of Lyme arthritis.

METHODS

Using cytometric bead array and flow cytometry techniques, chemokine and cytokine levels were determined in 65 synovial fluid (SF) samples and 7 synovial tissue (ST) samples from 17 patients with antibiotic-responsive Lyme arthritis and 35 patients with antibiotic-refractory Lyme arthritis seen during the past 18 years. In the ST samples, expression of chemokine receptors was measured using immunohistochemistry.

RESULTS

Before or during antibiotic therapy, when the majority of patients had positive polymerase chain reaction (PCR) results for Borrelia burgdorferi DNA, SF from patients with antibiotic-refractory arthritis contained exceptionally high levels of Th1 chemoattractants and cytokines, particularly CXCL9 and interferon-gamma (IFNgamma). Compared with the patients whose arthritis was responsive to antibiotic treatment, those with antibiotic-refractory arthritis had significantly higher levels of CXCL9 and CXCL10 (both P<or=0.001) and CCL3, CCL4, <em>CXCL8</em>, IFNgamma, tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-6 (all P<or=0.01). During the post-antibiotic period, when the results of PCR for B burgdorferi DNA in SF and ST were uniformly negative, patients with antibiotic-refractory arthritis continued to exhibit high SF and ST levels of these chemokines and cytokines. In addition, synovial samples showed marked expression of the receptors for T cell or macrophage chemokines, CXCR3 and CCR5.

CONCLUSIONS

Patients with antibiotic-refractory Lyme arthritis have high synovial fluid levels of proinflammatory chemokines and cytokines, especially CXCL9 and IFNgamma, throughout the illness. Thus, even when antibiotic treatment reduces or completely clears the infection in these patients, the inflammatory response in synovium persists.

We have recently shown that granulocyte-colony-stimulating factor (G-CSF)- and interferon-gamma (IFN-gamma)-activated human neutrophils accumulate and release remarkable amounts of soluble B-lymphocyte stimulator (BLyS) in vitro. In this study, we provide evidence that neutrophils migrating into skin window exudates (SWEs) developed in healthy volunteers and in patients with rheumatoid arthritis (RA), synthesized, and released BLyS in response to locally produced G-CSF. Accordingly, the concentrations of soluble BLyS in SWEs were significantly more elevated than in serum. Because the levels of SWE BLyS, but not SWE G-CSF, were higher in patients with RA than in healthy subjects, we examined the effect of CXCL8/IL-8, C5a, and other proinflammatory mediators that dramatically accumulate in RA SWEs and in inflamed synovial fluids. We show that CXCL1/GROalpha, CXCL8/IL-8, C5a, immune complexes, tumor necrosis factor-alpha (TNF-alpha), leukotriene B4, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and lipopolysaccharide (LPS), which by themselves do not induce BLyS de novo synthesis, act as potent secretagogues for BLyS, which is mainly stored in Golgi-related compartments within G-CSF-treated neutrophils, as determined by immunogold electron microscopy. This action is pivotal in greatly amplifying neutrophil-dependent BLyS release in SWEs of patients with RA compared with healthy subjects. Collectively, our data uncover a novel mechanism that might dramatically exacerbate the release of BLyS by neutrophils during pathologic inflammatory responses.

Alveolar macrophages (AM) are one of the key cell types for initiating inflammatory and immune responses to influenza virus in the lung. However, the genome-wide changes in response to influenza infection in AM have not been defined. We performed gene profiling of human AM in response to H1N1 influenza A virus PR/8 using Affymetrix HG-U133 Plus 2.0 chips and verified the changes at both mRNA and protein levels by real-time RT-PCR and ELISA. We confirmed the response with a contemporary H3N2 influenza virus A/New York/238/2005 (NY/238). To understand the local cellular response, we also evaluated the impact of paracrine factors on virus-induced chemokine and cytokine secretion. In addition, we investigated the changes in the expression of macrophage receptors and uptake of pathogens after PR/8 infection. Although macrophages fail to release a large amount of infectious virus, we observed a robust induction of type I and type III interferons and several cytokines and chemokines following influenza infection. CXCL9, 10, and 11 were the most highly induced chemokines by influenza infection. UV-inactivation abolished virus-induced cytokine and chemokine response, with the exception of CXCL10. The contemporary influenza virus NY/238 infection of AM induced a similar response as PR/8. Inhibition of TNF and/or IL-1β activity significantly decreased the secretion of the proinflammatory chemokines CCL5 and CXCL8 by over 50%. PR/8 infection also significantly decreased mRNA levels of macrophage receptors including C-type lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), and CD36, and reduced uptake of zymosan. In conclusion, influenza infection induced an extensive proinflammatory response in human AM. Targeting local components of innate immune response might provide a strategy for controlling influenza A infection-induced proinflammatory response in vivo.

This study investigated the role of heme oxygenase (HO)-1 in the cardiac tissue injury of acute ischemia/reperfusion (I/R) in diabetic streptozotocin (STZ)-induced hyperglycemic rats. The effects of 1) hemin, an inducer of HO expression and activity, and 2) zinc protoporphyrin IX (ZnPP-IX), an inhibitor of HO activity, have also been investigated on the tissue injury by I/R and some mediators released in these circumstances. STZ hyperglycemic rats had impaired levels of HO-1 within the cardiac tissue and increased myocardial infarct size (IS) following I/R, as compared with the nondiabetic rats. In these rats, administration of hemin 4 mg/kg 18 h before I/R increases the levels of HO-1 within the tissue. However, the values of HO-1 assayed in these circumstances were significantly lower (P < 0.01) than those assayed in nondiabetic animals subjected to the same procedures; IS was much more extended (P < 0.01) than in the parent nondiabetic group. STZ hyperglycemic rats also predisposed the heart to produce high levels of the cytokines interleukin (IL)-1beta and CXCL8. Subsequent I/R further increased (P < 0.01) the cytokine production, an effect partly prevented by hemin treatment. This recovered the huge number of infiltrated polymorphonuclear (PMN) leukocytes within the cardiac tissue associated with the STZ hyperglycemic state and I/R damage.

The Duffy blood group Ag (dfy) binds selective CXC and CC chemokines at high affinity and is expressed on erythrocytes and endothelial cells. However, it does not transmit a signal via G proteins, as occurs with other seven-transmembrane receptors. We hypothesized that dfy functions as a chemokine reservoir and regulates inflammation by altering soluble chemokine concentrations in the blood and tissue compartments. We determined whether Duffy Ag "loss-of-function" phenotypes (human and murine) are associated with alterations in plasma chemokine concentrations during the innate inflammatory response to LPS. Plasma CXCL8 and CCL2 concentrations from humans homozygous for the GATA-1 box polymorphism, a dfy polymorphism that abrogates erythrocyte chemokine binding, were higher than in heterozygotes following LPS stimulation of their whole blood in vitro. Similarly, dfy(-/-) mice showed higher plasma MIP-2 concentrations than dfy(+/+) mice following LPS stimulation of whole blood in vitro. We then determined the relative contributions of erythrocyte and endothelial Duffy Ag in modifying chemokine concentrations and neutrophil recruitment in the lungs following intratracheal LPS administration in dfy(-/-) and dfy(+/+) mice reconstituted with dfy(-/-) or dfy(+/+) marrow. Mice lacking endothelial dfy expression had higher MIP-2 and keratinocyte chemoattractant concentrations in the airspaces. Mice lacking erythrocyte dfy had higher MIP-2 and keratinocyte chemoattractant concentrations in the lung tissue vascular space, but lower plasma chemokine concentrations associated with attenuated neutrophil recruitment into the airspaces. These data indicate that dfy alters soluble chemokine concentrations in blood and local tissue compartments and enhances systemic bioavailability of chemokines produced during local tissue inflammation.

Neutrophils, historically known for their involvement in acute inflammation, are also targets for infection by many different DNA and RNA viruses. However, the mechanisms by which they recognize and respond to viral components are poorly understood. Polyinosinic:polycytidylic acid (poly(I:C)) is a synthetic mimetic of viral dsRNA that is known to interact either with endosomal TLR3 (not expressed by human neutrophils) or with cytoplasmic RNA helicases such as melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). In this study, we report that intracellularly administered poly(I:C) stimulates human neutrophils to specifically express elevated mRNA levels encoding type I IFNs, immunoregulatory cytokines, and chemokines, such as TNF-alpha, IL-12p40, CXCL10, CXCL8, CCL4, and CCL20, as well as classical IFN-responsive genes (IRG), including IFIT1 (IFN-induced protein with tetratricopeptide repeats 1)/IFN-stimulated gene (ISG)56, G1P2/ISG15, PKR (dsRNA-dependent protein kinase), and IFN-regulatory factor (IRF)7. Investigations into the mechanisms whereby transfected poly(I:C) promotes gene expression in neutrophils uncovered a crucial involvement of the MAPK-, PKR-, NF-kappaB-, and TANK (TNF receptor-associated NF-kappaB kinase)-binding kinase (TBK1)/IRF3-signaling transduction pathways, as illustrated by the use of specific pharmacological inhibitors. Consistent with the requirement of the cytoplasmic dsRNA pathway for antiviral signaling, human neutrophils were found to constitutively express significant levels of both MDA5 and RIG-I, but not TLR3. Accordingly, neutrophils isolated from MDA5-deficient mice had a partial impairment in the production of IFN-beta and TNF-alpha upon infection with encephalomyocarditis virus. Taken together, our data demonstrate that neutrophils are able to activate antiviral responses via helicase recognition, thus acting at the frontline of immunity against viruses.

Activation of the NADPH oxidase homolog dual oxidase 1 (DUOX1) within the airway epithelium represents a key mechanism of innate airway host defense, through enhanced production of H2O2, which mediates cellular signaling pathways that regulate the production of various inflammatory mediators. Production of the CXC chemokine interleukin (IL)-8/CXCL8 forms a common epithelial response to many diverse stimuli, including bacterial and viral triggers, environmental oxidants, and other biological mediators, suggesting the potential involvement of a common signaling pathway that may involve DUOX1-dependent H2O2 production. Following previous reports showing that DUOX1 is activated by extracellular ATP and purinergic receptor stimulation, this study demonstrates that airway epithelial IL-8 production in response to several bacterial stimuli involves ATP release and DUOX1 activation. ATP-mediated DUOX1 activation resulted in the activation of ERK1/2 and NF-kappaB pathways, which was associated with epidermal growth factor receptor (EGFR) ligand shedding by ADAM17 (a disintegrin and metalloproteinase-17). Although ATP-mediated ADAM17 activation and IL-8 release were not prevented by extracellular H2O2 scavenging by catalase, these responses were attenuated by intracellular scavengers of H2O2 or related oxidants, suggesting an intracellular redox signaling mechanism. Both ADAM17 activation and IL-8 release were suppressed by inhibitors of EGFR/ERK1/2 signaling, which can regulate ADAM17 activity by serine/threonine phosphorylation. Collectively, our results indicate that ATP-mediated DUOX1 activation represents a common response mechanism to several environmental stimuli, involving H2O2-dependent EGFR/ERK activation, ADAM17 activation, and EGFR ligand shedding, leading to amplified epithelial EGFR activation and IL-8 production.

Tumor-associated macrophages (TAMs) can promote cancer initiation and progression by releasing cytokines. Previously, we have found the density of TAMs correlated with lymph node metastasis in papillary thyroid carcinoma (PTC). However, the mechanisms of how TAMs promote PTC progression remain unclear. In this study, we first showed that the TAMs density in the tumor core was associated with progressive PTC features and TAMs conditioned medium enhanced PTC cells invasion. Cytokine profiling identified a mixed M1/M2 phenotype and CXCL8 was the most consistently abundant cytokine in PTC-derived TAMs. CXCL8 receptors, CXCR1 and CXCR2, were positively stained in PTC cell lines and tissues, though no association with lymph node metastasis or extrathyroid extension. PTC cell invasion was abrogated by anti-CXCL8-neutralizing antibody, whereas addition of exogenous recombinant human CXCL8 enhanced the invasiveness. More importantly, CXCL8 promoted PTC metastasis in vivo. No difference was found for TAMs-derived CXCL8 expression in patients with and without lymph node metastasis or extrathyroid extension. These findings indicated that TAMs may facilitate PTC cell metastasis through CXCL8 and its paracrine interaction with CXCR1/2.

BACKGROUND

The failure of neutrophils to migrate to an infection focus during severe sepsis is an important determinant of the inability of a host to deal with an infectious insult. Our laboratory has shown that inducible nitric oxide synthase (iNOS) induction and NO production contribute to the failure of neutrophils to migrate in the context of sepsis.

OBJECTIVE

We investigated whether CXCR2 expression contributed to the failure of neutrophils to migrate during severe sepsis and the role of NO in modulating CXCR2 expression on neutrophils in mice subjected to nonsevere (NS) or severe (S) cecal ligation and puncture (CLP).

RESULTS

Neutrophil migration to the infection focus was deficient in S-CLP mice, a phenomenon prevented by pharmacologic (aminoguanidine, l-canavanine) or genetic (iNOS gene deletion) inhibition of iNOS. The expression of CXCR2 on neutrophils from S-CLP mice was significantly reduced when compared with neutrophils from NS-CLP or sham-operated mice. CXCR2 expression was reestablished by pharmacologic and genetic inhibition of iNOS. Immunofluorescence and confocal analysis revealed that iNOS blockade reduced neutrophil CXCR2 internalization. Adhesion and emigration of neutrophils in macrophage inflammatory protein-2-stimulated mesentery microcirculation were reduced in S-CLP mice, compared with NS-CLP mice, and reestablished by pretreatment with aminoguanidine or l-canavanine. The NO donor S-nitroso-N-acetyl-d,l-penicillamine inhibited CXCL8-induced human neutrophil chemotaxis and CXCR2 expression on human and murine neutrophils.

CONCLUSIONS

These results highlight evidences that the failure of neutrophils to migrate to an infection focus during severe sepsis is associated with excessive NO production and NO-dependent regulation of the expression of CXCR2 on the neutrophil surface.

Constitutive activation of nuclear factor (NF)-kappaB is linked with the intrinsic resistance of androgen-independent prostate cancer (AIPC) to cytotoxic chemotherapy. Interleukin-8 (CXCL8) is a transcriptional target of NF-kappaB whose expression is elevated in AIPC. This study sought to determine the significance of CXCL8 signaling in regulating the response of AIPC cells to oxaliplatin, a drug whose activity is reportedly sensitive to NF-kappaB activity. Administration of oxaliplatin to PC3 and DU145 cells increased NF-kappaB activity, promoting antiapoptotic gene transcription. In addition, oxaliplatin increased the transcription and secretion of CXCL8 and the related CXC-chemokine CXCL1 and increased the transcription and expression of CXC-chemokine receptors, especially CXC-chemokine receptor (CXCR) 2, which transduces the biological effects of CXCL8 and CXCL1. Stimulation of AIPC cells with CXCL8 potentiated NF-kappaB activation in AIPC cells, increasing the transcription and expression of NF-kappaB-regulated antiapoptotic genes of the Bcl-2 and IAP families. Coadministration of a CXCR2-selective antagonist, AZ10397767 (Bioorg Med Chem Lett 18:798-803, 2008), attenuated oxaliplatin-induced NF-kappaB activation, increased oxaliplatin cytotoxicity, and potentiated oxaliplatin-induced apoptosis in AIPC cells. Pharmacological inhibition of NF-kappaBorRNA interference-mediated suppression of Bcl-2 and survivin was also shown to sensitize AIPC cells to oxaliplatin. Our results further support NF-kappaB activity as an important determinant of cancer cell sensitivity to oxaliplatin and identify the induction of autocrine CXCR2 signaling as a novel mode of resistance to this drug.

BACKGROUND

Receptor antagonists that block the binding of chemokines such as CXCL8 (IL-8) are effective in animals models of neutrophil-mediated inflammation. It has been hypothesized that selective inhibition of neutrophil trafficking and activation may be a useful adjunct for the treatment of inflammatory airway diseases such as chronic obstructive pulmonary disease or cystic fibrosis. A CXCR1/2 receptor antagonist has shown activity in an ozone challenge model in humans.

CONCLUSIONS

SB-656933, a selective CXCR2 antagonist, is safe and well-tolerated at single doses and is shown to inhibit agonist (CXCL1)-mediated expression of the CD11b on peripheral blood neutrophils as well as ozone-induced airway neutrophilia in healthy subjects.

OBJECTIVE

To determine the safety and tolerability of a novel selective CXCR2 antagonist and assess its pharmacodynamic effects using measures of neutrophil activation and function, including CD11b expression in whole blood and ozone-induced airway inflammation in healthy subjects.

METHODS

Flow cytometric determination of ex vivo CXCL1-induced CD11b expression on peripheral blood neutrophils was performed following single dose oral administration of SB-656933 (dose range 2-1100 mg). A subsequent randomized study (placebo, 50 mg and 150 mg) was performed to explore the dose-response for ozone-induced airway inflammation, as measured by sputum biomarkers.

RESULTS

Oral administration of SB-656933 resulted in significant inhibition of CXCL1-induced CD11b expression on peripheral blood neutrophils at single doses greater than or equal to 50 mg. Maximum inhibition (70%) relative to placebo was observed following administration of SB-656933 400 mg (95% CI 60%, 77%). This was sustained up to a dose of 1100 mg. Single doses of SB-656933 reduced ozone-induced airway inflammation in a dose-dependent manner. Relative to placebo, there were 55% (95% CI 20%, 75%) and 74% (95% CI 55%, 85%) fewer neutrophils in the sputum of subjects after a single dose of 50 mg or 150 mg, respectively. There was a corresponding reduction in myeloperoxidase concentrations in the sputum supernatant of 32.8% (95% CI 9.2, 50.3) and 50.5% (95% CI 33.3, 63.3). SB-656933 was safe and well-tolerated at all doses.

CONCLUSIONS

SB-656933 is a CXCR2 antagonist that demonstrates dose-dependent effects on neutrophil activation and recruitment within a well-tolerated dose range. These data suggest that SB-656933 may be an effective agent in neutrophil-predominant diseases.

Interleukin-17 (IL-17) is a CD4 T cell-derived proinflammatry and proangiogenic cytokine. In this study, we investigated the effects of this cytokine on vascular endothelial cell growth induced by a well-known direct angiogenic factor bFGF, HGF, VEGF, CXCL5/ENA-78 or CXCL8/IL-8. While a wide range of doses of IL-17 alone did not show the ability to stimulate the growth of human dermal microvascular endothelial cells (HMVECs), bFGF, HGF, VEGF, CXCL5 or CXCL8 significantly induced the growth of HMVECs in vitro. When bFGF and IL-17 were used in combination, 10 or 100 ng/ml IL-17 enhanced 10 ng/ml bFGF-induced growth of HMVECs. Similarly, when HGF and IL-17 were combined together, 10 or 100 ng/ml IL-17 potentiated 10 ng/ml HGF-induced growth of HMVECs. When VEGF and IL-17 were used together, 10 ng/ml IL-17 did not significantly enhance 10 ng/ml VEGF-induced growth, whereas 100 ng/ml IL-17 clearly promoted 10ng/ml VEGF-mediated proliferation of HMVECs. On the contrary, IL-17 did not augment CXCL5- and CXCL8-mediated growth. These results indicate that IL-17 itself does not have the capability to stimulate the growth of vascular endothelial cells, whereas IL-17 is able to selectively enhance the mitogenic activity of bFGF, HGF, and VEGF for vascular endothelial cells. Our findings also suggest that IL-17 may promote bFGF-, HGF- and VEGF-mediated angiogenesis through enhancing bFGF-, HGF- and VEGF-induced growth of vascular endothelial cells.

Within the brain, quinolinic acid (QUIN) is an important neurotoxin, especially in AIDS dementia complex (ADC). Its production by monocytic lineage cells is increased in the context of inflammation. However, it is not known whether QUIN promotes inflammation. Astrocytes are important in immunoregulation within the brain and so we chose to examine the effects of QUIN on the astrocyte. Using purified primary human fetal astrocyte cultures, we determined chemokine production using ELISA assays and RT-PCR and chemokine receptor expression using immunocytochemistry and RT-PCR with QUIN in comparison to TNFalpha, IL-1beta, and IFNgamma. We found that QUIN induces astrocytes to produce large quantities of MCP-1 (CCL2) and lesser amounts of RANTES (CCL5) and IL-8 (CXCL8). QUIN also increases SDF-1alpha (CXCL12), HuMIG (CXCL9), and fractalkine (CX(3)CL1) mRNA expression. Moreover, QUIN leads to upregulation of the chemokine receptor expression of CXCR4, CCR5, and CCR3 in human fetal astrocytes. Most of these effects were comparable to those induced by TNFalpha, IL-1beta, and IFNgamma. The present work represents the first evidence that QUIN induces chemokine and chemokine receptor expression in astrocytes and is at least as potent as classical mediators such as inflammatory cytokines. These results suggest that QUIN may be critical in the amplification of brain inflammation, particularly in ADC.

BACKGROUND

Rheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmune disease that leads to progressive cartilage destruction. Advances in the treatment of RA-related destruction of cartilage require profound insights into the molecular mechanisms involved in cartilage degradation. Until now, comprehensive data about the molecular RA-related dysfunction of chondrocytes have been limited. Hence, the objective of this study was to establish a standardized in vitro model to profile the key regulatory molecules of RA-related destruction of cartilage that are expressed by human chondrocytes.

METHODS

Human chondrocytes were cultured three-dimensionally for 14 days in alginate beads and subsequently stimulated for 48 hours with supernatants from SV40 T-antigen immortalized human synovial fibroblasts (SF) derived from a normal donor (NDSF) and from a patient with RA (RASF), respectively. To identify RA-related factors released from SF, supernatants of RASF and NDSF were analyzed with antibody-based protein membrane arrays. Stimulated cartilage-like cultures were used for subsequent gene expression profiling with oligonucleotide microarrays. Affymetrix GeneChip Operating Software and Robust Multi-array Analysis (RMA) were used to identify differentially expressed genes. Expression of selected genes was verified by real-time RT-PCR.

RESULTS

Antibody-based protein membrane arrays of synovial fibroblast supernatants identified RA-related soluble mediators (IL-6, CCL2, CXCL1-3, CXCL8) released from RASF. Genome-wide microarray analysis of RASF-stimulated chondrocytes disclosed a distinct expression profile related to cartilage destruction involving marker genes of inflammation (adenosine A2A receptor, cyclooxygenase-2), the NF-kappaB signaling pathway (toll-like receptor 2, spermine synthase, receptor-interacting serine-threonine kinase 2), cytokines/chemokines and receptors (CXCL1-3, CXCL8, CCL20, CXCR4, IL-1beta, IL-6), cartilage degradation (matrix metalloproteinase (MMP)-10, MMP-12) and suppressed matrix synthesis (cartilage oligomeric matrix protein, chondroitin sulfate proteoglycan 2).

CONCLUSIONS

Differential transcriptome profiling of stimulated human chondrocytes revealed a disturbed catabolic-anabolic homeostasis of chondrocyte function and disclosed relevant pharmacological target genes of cartilage destruction. This study provides comprehensive insight into molecular regulatory processes induced in human chondrocytes during RA-related destruction of cartilage. The established model may serve as a human in vitro disease model of RA-related destruction of cartilage and may help to elucidate the molecular effects of anti-rheumatic drugs on human chondrocyte gene expression.

Substance P analogues, including [D-Arg(1),D-Trp(5,7,9),Leu(11)]SP (SPA) are broad-spectrum G protein-coupled receptor (GPCR) antagonists that have potential antitumorigenic activities, although the mechanism(s) are not completely understood. Here, we examined the effects of SPA in ductal pancreatic cancers that express multiple GPCRs for mitogenic agonists and also produce proangiogenic chemokines. Using HPAF-II, a well-differentiated pancreatic cancer cell line as our model system, we showed that SPA inhibited multiple neuropeptide-induced Ca(2+) mobilization, DNA synthesis, and anchorage-independent growth in vitro. SPA also significantly attenuated the growth of HPAF-II tumor xenografts in nude mice beyond the treatment period. Interestingly, SPA markedly increased apoptosis but moderately decreased proliferation marker, Ki-67 in the tumor xenografts implying additional mechanism(s) for the significant growth inhibitory effect observed in vivo. HPAF-II cells express ELR(+) CXC chemokines, including IL-8/CXCL8, which bind to CXCR2 (a member of GPCR superfamily) and promote angiogenesis in multiple cancers, including pancreatic cancer. SPA inhibited CXCR2-mediated Ca(2+) mobilization and blocked specifically IL-8/CXCL8-induced angiogenesis in rat corneal micropocket assay in vivo. A salient feature of the results presented here is that SPA markedly reduced tumor-associated angiogenesis in the HPAF-II xenografts in vivo. Our results show that SPA, a broad-spectrum GPCR antagonist attenuates tumor growth in pancreatic cancer via a dual mechanism involving both the antiproliferative and antiangiogenic properties. We conclude that this novel dual-inhibitory property of SPA could be of significant therapeutic value in pancreatic cancer, when used in combination with other antiproliferative and/or antiangiogenic agents.

Hepatitis C virus (HCV) infection is frequently complicated by glomerulonephritis with immune complexes containing viral RNA. We examined the potential influence of Toll-like receptors (TLRs), specifically TLR3 recognition of viral dsRNA exemplified by polyriboinosinic:polyribocytidylic acid [poly(I:C) RNA]. Normal human kidney stained positive for TLR3 on mesangial cells (MCs), vascular smooth muscle cells, and collecting duct epithelium. Cultured MCs have low TLR3 mRNA levels with predominant intracellular protein localization, which was increased by tumor necrosis factor-alpha, interleukin (IL)-1beta, interferon (IFN)-gamma, and the TLR3 ligand poly(I:C) RNA. Poly(I:C) RNA stimulation of MCs increased mRNA and protein synthesis of IL-6, IL-1beta, M-CSF, IL-8/CXCL8, RANTES/CCL5, MCP-1/CCL2, and ICAM-I; it also increased anti-proliferative and proapoptotic effects, the latter of which was decreased by inhibiting caspase-8. In microdissected glomeruli of normal and non-HCV membranoproliferative glomerulonephritis biopsies, TLR3 mRNA expression was low. In contrast TLR3 mRNA expression was significantly increased in hepatitis C-positive glomerulonephritis and was associated with enhanced mRNA for RANTES/CCL5 and MCP-1/CCL2. We hypothesize that immune complexes containing viral RNA activate mesangial TLR3 during HCV infection, thereby contributing to chemokine/cytokine release and effecting proliferation and apoptosis. Thus, TLR3 expression on renal cells, and especially MCs, may establish a link between viral infections and glomerular diseases.

Theiler's murine encephalomyelitis virus (TMEV) infection in the central nervous system (CNS) induces a demyelinating disease similar to human multiple sclerosis. TMEV infection results in activation of various chemokine and cytokine genes that are important in the initiation of an inflammatory response. We have previously shown that the production of these chemokines and cytokines in astrocytes is induced via the NF-kappaB pathway following TMEV and Coxsackie virus infection. In this study, we investigated whether the NF-kappaB-dependent inflammatory responses after TMEV infection is triggered through TLR3 and/or TLR7. The activation of NF-kappaB or IRF/ISRE, as well as the production of both MCP-1/CCL2 and IL-8/CXCL8, was observed in only TLR3-transfected HEK 293 cells, but not in TLR7-tranfected cells. The potential involvement of TLR3 in mouse embryonic fibroblasts and primary astrocytes was further investigated following transfection with wildtype or dominant negative form of TLRs and MyD88, as well as astrocytes from TLR3- and MyD88-deficient mice. Similarly, the activation of transcription factors and chemokine genes is induced in these mouse cells through primarily TLR3 signaling pathway, but not TLR7 or other MyD88-mediated pathways following TMEV infection. However, the TLR3-mediated cellular activation does not appear to affect the level of viral replication in astrocytes. These results strongly suggest that TLR3-signaling by TMEV alone is sufficient to induce the initial inflammatory cytokine responses that could be very important for the outcome of virus-induced encephalitis and/or demyelinating diseases, such as multiple sclerosis.

Chemokines regulate leukocyte migration during physiological and pathological conditions. They exert their biological activity through interaction with 7-transmembrane spanning G protein-coupled receptors (GPCR) and are presented on glycosaminoglycans (GAG) linked to endothelial cell layers. Specific chemokines and chemokine receptors affect angiogenesis or are targets for viral mimicry, e.g. by human immunodeficiency virus (HIV). Several enzymes, in particular proteases, have been described to process chemokines at specific sites generating chemokine isoforms that were also identified from natural sources. For some chemokines, e.g. CXCL8 and CCL3L1, posttranslational modification results in enhanced biological activity. For CXCL7 and CCL14 truncation is even mandatory for receptor signaling and chemotactic properties. The activity of many other chemokines is down-regulated by processing and receptor antagonists are generated, e.g. for truncated CCL8 and CCL11. Moreover, some processed chemokines such as CCL5(3-68) show enhanced affinity for one receptor (CCR5) and reduced interaction with other receptors (CCR1 and CCR3) resulting in differential changes in leukocyte response. These posttranslational mechanisms, in addition to gene duplication, transcriptional and translational regulation of chemokine ligand and receptor expression, GAG binding properties, expression of "silent" receptors and synergistic interaction between chemokines, modulate chemokine activity in a complex manner. This report reviews current understanding on the regulation of the chemokine network through posttranslational modification and its consequences for leukocyte migration, angiogenesis and protection against viral infection.