Ramses Ilarraza

Virus-induced eosinophil mediator release requires antigen-presenting and CD4+ T cells

BACKGROUND The most frequent trigger of asthma exacerbation is infection with common airway viruses; however, the precise mechanism regulating such severe reactions is not understood. The presence of airway eosinophil products is a unique feature detected in asthmatic airways. Using an animal model, we previously demonstrated that T cells play an important role in regulating an eosinophil-dependant pathway of virus-induced airway hyperreactivity. We hypothesize that human eosinophils respond to viruses, although only after interaction with T cells. OBJECTIVES We sought to determine whether eosinophils can respond to airway viruses in vitro and determine the mechanism of response. METHODS An in vitro coculture model of human eosinophils, antigen-presenting cells, and T cells was used with parainfluenza virus, respiratory syncytial virus, or rhinovirus. We measured release of eosinophil peroxidase (EPO) in concert with T-cell proliferation, cytokine release, and changes in T-cell phenotype. RESULTS The viruses induced release of EPO when coincubated in the presence of antigen-presenting cells (dendritic cells or macrophages) and T cells. Virus-mediated release was associated with proliferation of CD3(+)CD4(+) T cells and release of cytokines. UV inactivation of the virus did not prevent virus-induced EPO release or T-cell proliferation. Proliferating CD4(+) T cells show increased expression of CD25 and CD45RO. CD8(+) T cells were not activated. CONCLUSION Virus-induced EPO release can occur in the context of antigen presentation to CD4(+) T cells.

Respiratory syncytial virus induces indoleamine 2,3‐dioxygenase activity: a potential novel role in the development of allergic disease

Infants that develop severe bronchiolitis due to respiratory syncytial virus (RSV) are at increased risk of developing asthma later in life. We investigated a potential immunological mechanism for the association between RSV and the development of allergic inflammation. The enzyme indoleamine 2,3‐dioxygenase (IDO) has been reported to induce selective apoptosis of T helper 1 (Th1) cells and contributed to Th2‐biased immune responses.

The effect of cationic charge on release of eosinophil mediators

BACKGROUND In patients with atopic diseases, cationic-charged eosinophil proteins are deposited in inflamed tissues. Although the role of cytokines in cell activation is well established, the presence of cationic-charged tissue can also be an important factor in inflammatory cell function. OBJECTIVES We sought to determine whether increased cationic charge seen in an atopic microenvironment plays a role in the activation of eosinophils. METHODS Human eosinophils were incubated with Sepharose beads coated with cationic or anionic compounds in the presence and absence of a cytokine cocktail (IL-3, IL-5, and GM-CSF) to simulate the milieu of inflammation. Eosinophil peroxidase and eosinophil-derived neurotoxin (EDN) release were compared with eosinophil morphology and expression of CD18, as determined by means of confocal microscopy. RESULTS Cytokines with positively charged beads caused greater eosinophil peroxidase release (lysine coated, 44.2 nmol/L; compound 48/80, 40.0 nmol/L; or EDN coated, 49.1 nmol/L) than cytokines alone (14.9 nmol/L). Beads coated with heparin, dextran sulfate, and aspartic acid did not show this effect. EDN release was also induced by lysine-coated beads with cytokines (67.1 ng/100 microL) and blocked by heparin. Eosinophil incubation with wortmannin, genistein, and the src kinase inhibitor PP1 blocked cationic signaling. Eosinophils adherent to cationic-charged beads but not anionic-charged beads show polarization of CD18 expression toward the beads surface. CONCLUSION Cationic-charged surfaces induce increased eosinophil mediator release by increasing the density of CD18 expression available at the target surface.

Connexin 43 expression on peripheral blood eosinophils: role of gap junctions in transendothelial migration.

Eosinophils circulate in the blood and are recruited in tissues during allergic inflammation. Gap junctions mediate direct communication between adjacent cells and may represent a new way of communication between immune cells distinct from communication through cytokines and chemokines. We characterized the expression of connexin (Cx)43 by eosinophils isolated from atopic individuals using RT-PCR, Western blotting, and confocal microscopy and studied the biological functions of gap junctions on eosinophils. The formation of functional gap junctions was evaluated measuring dye transfer using flow cytometry. The role of gap junctions on eosinophil transendothelial migration was studied using the inhibitor 18-a-glycyrrhetinic acid. Peripheral blood eosinophils express Cx43 mRNA and protein. Cx43 is localized not only in the cytoplasm but also on the plasma membrane. The membrane impermeable dye BCECF transferred from eosinophils to epithelial or endothelial cells following coculture in a dose and time dependent fashion. The gap junction inhibitors 18-a-glycyrrhetinic acid and octanol did not have a significant effect on dye transfer but reduced dye exit from eosinophils. The gap junction inhibitor 18-a-glycyrrhetinic acid inhibited eosinophil transendothelial migration in a dose dependent manner. Thus, eosinophils from atopic individuals express Cx43 constitutively and Cx43 may play an important role in eosinophil transendothelial migration and function in sites of inflammation.

Tumor necrosis factor regulates NMDA receptor-mediated airway smooth muscle contractile function and airway responsiveness

We have shown that N-methyl-d-aspartate receptors (NMDA-Rs) are receptor-operated calcium entry channels in human airway smooth muscle (HASM) during contraction. Tumor necrosis factor (TNF) augments smooth muscle contractility by influencing pathways that regulate intracellular calcium flux and can alter NMDA-R expression and activity in cortical neurons and glial cells. We hypothesized that NMDA-R-mediated Ca(2+) and contractile responses of ASM can be altered by inflammatory mediators, including TNF. In cultured HASM cells, we assessed TNF (10 ng/ml, 48 h) effect on NMDA-R subunit abundance by quantitative PCR, confocal imaging, and immunoblotting. We observed dose- and time-dependent changes in NMDA-R composition: increased obligatory NR1 subunit expression and altered regulatory NR2 and inhibitory NR3 subunits. Measuring intracellular Ca(2+) flux in Fura-2-loaded HASM cultures, we observed that TNF exposure enhanced cytosolic Ca(2+) mobilization and changed the temporal pattern of Ca(2+) flux in individual myocytes induced by NMDA, an NMDA-R selective analog of glutamate. We measured airway responses to NMDA in murine thin-cut lung slices (TCLS) from allergen-naive animals and observed significant airway contraction. However, NMDA acted as a bronchodilator in TCLS from house dust mice-challenged mice and in allergen-naive TCLS subjected to TNF exposure. All contractile or bronchodilator responses were blocked by a selective NMDA-R antagonist, (2R)-amino-5-phosphonopentanoate, and bronchodilator responses were prevented by N(G)-nitro-l-arginine methyl ester (nitric oxide synthase inhibitor) or indomethacin (cyclooxygenase inhibitor). Collectively, we show that TNF augments NMDA-R-mediated Ca(2+) mobilization in HASM cells, whereas in multicellular TCLSs allergic inflammation and TNF exposure leads to NMDA-R-mediated bronchodilation. These findings reveal the unique contribution of ionotrophic NMDA-R to airway hyperreactivity.

Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin‐dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT‐PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet‐activating factor, and secretory IgA‐Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock‐down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin‐4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist‐induced degranulation.

Human dendritic cells promote an antiviral immune response when stimulated by CVT-E002.

Objectives  There is interest in developing new compounds to enhance the immune response to airway virus infections. CVT‐E002 is a patented ginseng extract shown to decrease symptoms of virus infection in clinical trials. We hypothesized that the mechanism for this antiviral effect could be through modulation of dendritic cells leading to enhanced T‐cell activation.

Compound CVT-E002 attenuates allergen-induced airway inflammation and airway hyperresponsiveness, in vivo

Immune modulation has been a sought after means of therapy for atopic diseases. CVT-E002 is an extract derived from North American Ginseng shown to promote T-helper-1-like responses. We determined what effect CVT-E002 could have in a mouse model of atopic asthma. We report that oral CVT-E002 inhibited the development of allergic airway inflammation and airway hyperresponsiveness. This correlated with an increased presence of interferon-γ in the lung, and also increased regulatory T cells and IL-10. The ability of CVT-E002 to induce regulatory T-cell development was also seen in human in vitro co-cultures.

Montelukast inhibits leukotriene stimulation of human dendritic cells in vitro.

Background: Leukotrienes are potent inflammatory mediators which modulate immune responses and induce bronchoconstriction in susceptible individuals. Montelukast (MK) is a leukotriene receptor (CysLT1) antagonist that has been shown to prevent exacerbation of asthma. Considering the plethora of potential cellular targets for MK, specific mechanisms for its therapeutic action are still not fully understood. In vitro, we determined whether human dendritic cell function could be affected by leukotriene C4 (LTC4) treatment and whether MK had potential in modulating this response. We also studied the effect of LTC4 in the context of response to an airway virus (respiratory syncytial virus, RSV). Methods: Human monocyte-derived dendritic cells (moDCs) exposed to LTC4, MK, or both, were cocultured with autologous T cells, with or without RSV. The effects of LTC4 and MK on cell function were determined by ELISA and proliferation assays. Results: Both moDCs and their precursors – monocytes – express LTC4 receptor CysLT1, making them potential targets for MK. moDCs cultured with LTC4 release the eosinophil chemoattractant RANTES (CCL5) and induce greater T cell proliferation. Both were blocked by the presence of MK. MK treatment, albeit anti-inflammatory, did not interfere with the moDC-dependent T cell-proliferative responses induced by RSV. Conclusions: LTC4, chronically present in the airways of asthma patients, could induce an exaggerated inflammatory response to airway infection via dendritic cell activation, which would be prevented by MK. Our study provides additional insight into the mechanisms of action of this leukotriene receptor antagonist.

Predictive Value of Matrix Metalloproteinases and Their Inhibitors for Mortality in Septic Patients: A Cohort Study

Purpose: Over 170 biomarkers are being investigated regarding their prognostic and diagnostic accuracy in sepsis in order to find new tools to reduce morbidity and mortality. Matrix metalloproteinases (MMPs) and their inhibitors have been recently studied as promising new prognostic biomarkers in patients with sepsis. This study is aimed at determining the utility of several cutoff points of these biomarkers to predict mortality in patients with sepsis. Materials and Methods: A multicenter, prospective, analytic cohort study was performed in the metropolitan area of Bucaramanga, Colombia. A total of 289 patients with sepsis and septic shock were included. MMP-9, MMP-2, tissue inhibitor of metalloproteinase 1 (TIMP-1), TIMP-2, TIMP-1/MMP-9 ratio, and TIMP-2/MMP-2 ratio were determined in blood samples. Value ranges were correlated with mortality to estimate sensitivity, specificity, positive predictive value, negative predictive value, and area under the receiving operating characteristic curve. Results: Sensitivity ranged from 33.3% (MMP-9/TIMP-1 ratio) to 60.6% (TIMP-1) and specificity varied from 38.8% (MMP-2/TIMP-2 ratio) to 58.5% (TIMP-1). As for predictive values, positive predictive value range was from 17.5% (MMP-9/TIMP-1 ratio) to 70.4% (MMP-2/TIMP-2 ratio), whereas negative predictive values were between 23.2% (MMP-2/TIMP-2 ratio) and 80.9% (TIMP-1). Finally, area under the curve scores ranged from 0.31 (MMP-9/TIMP-1 ratio) to 0.623 (TIMP-1). Conclusion: Although TIMP-1 showed higher sensitivity, specificity, and negative predictive value, with a representative population sample, we conclude that none of the evaluated biomarkers had significant predictive value for mortality.