Yingqi Wu

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.

Eosinophils in human oral squamous carcinoma; role of prostaglandin D2

Eosinophils are often predominant inflammatory leukocytes infiltrating oral squamous carcinoma (OSC) sites. Prostaglandins are secreted by oral carcinomas and may be involved in eosinophil infiltration. The objective of this study was to determine the factors contributing to eosinophil migration and potential anti-neoplastic effects on OSC. Eosinophil degranulation was evaluated by measuring release of eosinophil peroxidase (EPO). Eosinophil chemotaxis towards OSC cells was assessed using artificial basement membrane. Eosinophil infiltration was prominent within the tissue surrounding the OSC tumor mass. We observed growth inhibition of the OSC cell line, SCC-9, during co-culture with human eosinophils, in vitro, which correlated with EPO activity that possesses growth inhibitory activity. The PGD2 synthase inhibitor, HQL-79, abrogated migration towards SCC-9. Our data suggest that OSC-derived PGD2 may play an important role via CRTH2 (the PGD2 receptor on eosinophils) in eosinophil recruitment and subsequent anti-tumor activity through the action of eosinophil cationic proteins.

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.

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.

Respiratory syncytial virus replication induces Indoleamine 2,3-dioxygenase (IDO) activation in human dendritic cells

Objective/purpose Induction of IDO in dendritic cells (DCs) depletes the essential amino acid, tryptophan, and generates a family of catabolites known as kynurenines (KYN). IDO activity is reported to have immunomodulatory effects, including the selective induction of apoptosis in T-helper 1 (Th1) lymphocytes, an effect not seen with Th2 cells that are dominant in allergic asthma. Infants hospitalized for RSV-related bronchiolitis have increased risk of developing asthma (48% vs. 8% in control). Induction of IDO activity by RSV may explain the link between RSV bronchiolitis and asthma pathogenesis. IDO is induced by various cytokines and a number of non-airway viruses; however, RSV has not yet been studied. We hypothesize that RSV induce IDO activation in human dendritic cells (DCs). Methods Primary human dendritic cells (DCs) were infected with sucrose gradient purified RSV with a multiplicity of infection (MOI) rate of 1.0. Flow cytometry and confocal microscopy were used to confirm infection. We measured KYN in culture media by a spectrophotometric method using Ehrlich reagent. We blocked RSV infection with the RSV-mAb, Palivizumab, and UV-inactivation to determine a role for infection. The potent competitive inhibitor of viral RNA polymerase, Ribavirin, was used to block RSV replication and protein synthesis. To evaluate dependency of RSV-induced IDO induction on different cell signaling pathways, we used a variety of specific inhibitors including MEK inhibitor I (120 nM), MEK inhibitor II (4 μM), SB202190 (p38MAPK, 3 μM), JNK inhibitor II (1 μM), IKK inhibitor II (Wedelolactone, 30 μM), IKK inhibitor III (BMS345541, 3 μM) and relevant negative controls.

Calcitriol reduces eosinophil cytolysis and release of cytotoxic granules in vitro

Background Epidemiological studies show a strong correlation between vitamin D deficiency and asthma severity. Genetic studies have identified vitamin D receptor polymorphisms as a risk factor for asthma in diverse human populations. Calcitriol (1,25-dihydroxyvitamin D3), the physiologically active metabolite of mammalian vitamin D, is able to modulate receptor and cytokine expression as well as cell differentiation and maturation of various leukocytes in an anti-inflammatory manner. Despite the role of eosinophils in allergic asthma pathology, little is known about the effects of calcitriol on eosinophil biology. Calcitriol has a direct modulatory effect on eosinophil survival and effector functions in vitro.