Induction of Interferon-γ and Tissue Inflammation by Overexpression of Eosinophil Cationic Protein in T Cells and Exosomes.

OBJECTIVE: T cells play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). Serum-derived exosomes are increased in SLE patients and are correlated with disease severity. This study was undertaken to investigate whether T cell-derived exosomal proteins play a role in SLE pathogenesis. METHODS: We characterized proteins in T cell-derived exosomes from SLE patients and healthy controls by MACSPlex exosome analysis and proteomics. To study the potential pathogenic functions of the exosomal protein identified, we generated and characterized T cell-specific transgenic mice that overexpressed that protein in T cells. RESULTS: We identified eosinophil cationic protein (ECP, also called human RNase III) as overexpressed in SLE T cell-derived exosomes. T cell-specific ECP-transgenic mice (n = 5 per group) displayed early induction of serum interferon-γ (IFNγ) levels (P = 0.062) and inflammation of multiple tissue types. Older T cell-specific ECP-transgenic mice (n = 3 per group) also displayed an increase in follicular helper T cell and plasma B cell numbers, and in autoantibody levels (P < 0.01). Single-cell RNA sequencing showed the induction of IFNγ messenger RNA (P = 2.2 × 10-13 ) and inflammatory pathways in ECP-transgenic mouse T cells. Notably, adoptively transferred ECP-containing exosomes stimulated serum autoantibody levels (P < 0.01) and tissue IFNγ levels in the recipient mice (n = 3 per group). The transferred exosomes infiltrated into multiple tissues of the recipient mice, resulting in hepatitis, nephritis, and arthritis. CONCLUSION: Our findings indicate that ECP overexpression in T cells or T cell-derived exosomes may be a biomarker and pathogenic factor for nephritis, hepatitis, and arthritis associated with SLE.

Eosinophil cationic protein mRNA expression in children with bronchial asthma.

Studies have shown that eosinophils are closely related to pathogenesis of bronchial asthma. Eosinophils release eosinophil cationic protein (ECP), which plays an important role in infection and allergic reactions. Serum ECP mRNA expression in children with bronchial asthma has not been adequately investigated. We analyzed serum ECP mRNA expression in 63 children with bronchial asthma and 21 healthy children by using reverse-transcriptase polymerase chain reaction to understand the role of ECP in children with bronchial asthma. The children with bronchial asthma were segregated into acute-phase and stable-phase groups, based on the severity of the illness. Serum ECP mRNA expression in children with bronchial asthma (0.375 ± 0.04) was significantly higher than that in healthy controls (0.20 ± 0.02; P < 0.05). Additionally, children in the acute-phase group showed higher ECP mRNA expression level (0.44 ± 0.06) than those in the stable-phase (0.31 ± 0.03) and healthy control groups (0.20 ± 0.02; P < 0.05), while the level in the stable-phase (0.31 ± 0.03) was markedly higher than that in the healthy control group (0.20 ± 0.02; P < 0.05). Detection of serum ECP mRNA expression level has possible applications in the diagnosis and treatment of children with bronchial asthma.

The production of the eosinophil proteins ECP and EPX/EDN are regulated in a reciprocal manner.

Previous studies showed that the biological activity and the eosinophil content of eosinophil cationic protein (ECP, RNase 3) are determined by single-nucleotide polymorphisms (SNPs) in the ECP (RNase3) gene. In this study, we report the prevalence of a common SNP in the eosinophil protein x/eosinophil-derived neurotoxin (EPX/EDN, RNase2) and the association with the cellular contents of EPX/EDN and ECP. The genes were sequenced and the EPX/EDN405(G>C) rs2013109 SNPs were also determined by TaqMan 5'nuclease allelic discrimination assay. ECP and EPX/EDN in purified eosinophils or in whole blood extracts were analysed by sensitive immunoassays. The study included 379 non-allergic and allergic subjects. The genotype prevalence of the EPX/EDN405(G>C) polymorphism was GG 59%, GC 36% and CC 6%. The cellular contents of ECP and EPX/EDN were related in a reciprocal fashion with the sums of the protein contents being constant. The contents were associated with the ECP562(G>C) rs2233860 and EPX/EDN405(G>C) gene polymorphisms. The cellular content of eosinophil peroxidase (EPO) was not associated with the ECP and EPX/EDN genotypes. The prevalence of the EPX/EDN405(G>C) genotypes and the contents of the proteins were similar in non-allergic and allergic subjects.The production and storage of the two ancestral proteins, ECP and EPX/EDN likely share common regulatory mechanisms, which result in opposing productions of the two proteins.

Bafetinib inhibits functional responses of human eosinophils in vitro.

Eosinophils play a prominent role in the process of allergic inflammation. Non-receptor associated Lyn tyrosine kinases generate key initial signals in eosinophils. Bafetinib, a specific Abl/Lyn tyrosine kinase inhibitor has shown a potent antiproliferative activity in leukemic cells, but its effects on eosinophils have not been reported. Therefore, we studied the effects of bafetinib on functional and mechanistic responses of isolated human eosinophils. Bafetinib was more potent than non-specific tyrosin kinase comparators genistein and tyrphostin inhibiting superoxide anion triggered by N-formyl-Met-Leu-Phe (fMLF; 100 nM) (-log IC50=7.25 ± 0.04 M; 6.1 ± 0.04 M; and 6.55 ± 0.03 M, respectively). Bafetinib, genistein and tyrphostin did not modify the [Ca(2+)]i responses to fMLF. Bafetinib inhibited the release of EPO induced by fMLF with higher potency than genistein and tyrphostin (-log IC50=7.24 ± 0.09 M; 5.36 ± 0.28 M; and 5.37 ± 0.19 M, respectively), and nearly suppressed LTC4, ECP and chemotaxis. Bafetinib, genistein and tyrphostin did not change constitutive apoptosis. However bafetinib inhibited the ability of granulocyte-monocyte colony-stimulating factor to prevent apoptosis. The activation of Lyn tyrosine kinase, p-ERK1/2 and p-38 induced by fMLF was suppressed by bafetinib and attenuated by genistein and tyrphostin. In conclusion, bafetinib inhibits oxidative burst and generation of inflammatory mediators, and reverses the eosinophil survival. Therefore, future anti-allergic therapies based on bafetinib, could help to suppress excessive inflammatory response of eosinophils at inflammatory sites.

Research resource: nuclear receptors as transcriptome: discriminant and prognostic value in breast cancer.

To identify biologically relevant groupings or clusters of nuclear receptors (NR) that are associated with breast neoplasia, with potentially diagnostic, discriminant or prognostic value, we quantitated mRNA expression levels of all 48 members of the human NR superfamily by TaqMan low-density array analysis in 116 curated breast tissue samples, including pre- and postmenopausal normal breast and both ERα(+) and ERα(-) tumor tissue. In addition, we have determined NR levels in independent cohorts of tamoxifen-treated ERα(+) and ERα(-) tissue samples. There were differences in relative NR mRNA expression between neoplastic and normal breast, and between ER(+) and ER(-) tumors. First, there is overexpression of the NUR77 subgroup and EAR2 in neoplastic breast. Second, we identify a signature of five NR (ERα, EAR2, NUR77, TRα, and RARγ) that classifies breast samples with more than 97% cross-validated accuracy into normal or cancer classes. Third, we find a novel negative association between five NR (TRß, NUR77, RORγ, COUP-TFII, and LRH1) and histological grade. Finally, four NR (COUP-TFII, TRß, PPARγ, and MR) are significant predictors of metastasis-free survival in tamoxifen-treated breast cancers, independent of ER expression. The present study highlights the discriminant and prognostic value of NR in breast cancer; identifies novel, clinically relevant, NR signatures; and highlights NR signaling pathways with potential roles in breast cancer pathophysiology and as new therapeutic targets.

Eosinophil cationic protein as a marker for assessing the efficacy of tacrolimus ophthalmic solution in the treatment of atopic keratoconjunctivitis.

PURPOSE: To examine the clinical efficacy and anti-inflammatory effects of tacrolimus eye drops; we studied the changes in clinical ocular findings and measured tear eosinophil cationic protein (ECP) levels of atopic keratoconjunctivitis (AKC) patients before and after the treatment. METHODS: Nine eyes of 9 patients (8 males, 1 female; mean age: 16.9 ± 11.4 years; range: 6-44 years) diagnosed with moderate or severe AKC disease were enrolled in this prospective study and treated with tacrolimus. All patients received 0.1% tacrolimus eye drops 2 times a day for 1 month. Tear samples were taken before and after treatment and ECP concentrations were obtained. Corneal fluorescein staining and conjunctival injection, edema, and papillary formation were graded on the recruitment day and one month later. Analysis of pre- and post-treatment findings was done using the Wilcoxon signed test. The ECP concentrations were correlated with the clinical signs using Spearman correlation tests. RESULTS: Post-treatment tear ECP levels were significantly reduced compared to the pre-treatment level. Clinical corneal scores also improved significantly after one month treatment with tacrolimus eye-drops. The mean conjunctival injection and conjunctival edema scores were significantly (p<0.05) decreased after the drug therapy. Strong positive linear correlations between ECP values and clinical signs were observed. Patients did not present side effects during the treatment with tacrolimus. CONCLUSIONS: In this pilot study, tacrolimus eye drops were found to reduce signs of AKC. ECP proved to correlate well with clinical signs of AKC.

Detection of Staphylococcus aureus in nasal tissue with peptide nucleic acid-fluorescence in situ hybridization.

BACKGROUND: Staphylococcus aureus (SA) in the nose can be a simple colonizer but also may create an intramucosal reservoir causing recurrent infections or can be a specific immune modulator through superantigenic mechanisms. Because the colonization rate of SA is high, but immunologic reactions causing chronic disease are less frequent, the purpose of this study was to identify the presence of intramucosal SA in healthy subjects and in patients with chronic rhinosinusitis (CRS) and to eventually relate those to the specific immunologic changes due to SA enterotoxins. METHODS: Nasal tissue was collected in 40 subjects (9 controls, 21 CRS patients with [CRSwNP], and 10 CRS patients without nasal polyps [CRSsNP]). Tissues were homogenized, and mediators and specific IgE-antibodies against SA enterotoxins (SAE-IgE) were measured using the UniCAP system. The tissue was analyzed for the presence of SA by the peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) technique (AdvanDx), and a semiquantitative scoring system was applied. Mann-Whitney exact test was used for statistical analysis. RESULTS: SA in the mucosal tissue was detected in a higher quantity among CRSwNP subjects with aspirin exacerbated respiratory disease (AERD) versus controls and CRSsNP (p=0.03). Among CRSwNP patients, Th2 markers (eosinophil cationic protein, p=0.006, and total IgE, p=0.004) were increased related to the SAE-IgE status but not related to the presence of SA in the tissue. CONCLUSION: This study describes the detection of SA within nasal tissue using the PNA-FISH technique. The presence of SA in the submucosa did not correlate with the amplification of the Th2-related inflammation typically found in CRSwNP patients, but this reaction is dependent on the formation of SAE-IgE within mucosal tissue. We also show, for the first time, that submucosal SA is a prevalent finding in CRSwNP patients with AERD.

Effects of daily intake of Lactobacillus paracasei strain KW3110 on Japanese cedar pollinosis.

Japanese cedar pollinosis is an important contributor to allergic rhinitis in Japan. Lactobacillus may be useful as an immunomodulator and is used widely as a foodstuff. The purpose of the study was to examine the effects of daily intake of the Lactobacillus paracasei strain KW3110 in patients with cedar pollinosis. The effects of daily intake of KW3110 in patients with cedar pollinosis were investigated in 126 patients who received KW3110 or a placebo in a double-blind study. The study began 1 month before the start of the pollen season and lasted for 3 months. A significant reduction of nasal symptoms and the serum level of eosinophil cationic protein and improvement of quality of life scores occurred in the patients who received KW3110 when pollen scattering was low. However, the effects were limited during the peak period of pollen scattering. Intake of KW3110 may reduce allergic inflammation, but the effect is limited.

Taurine chloramine inhibits functional responses of human eosinophils in vitro.

BACKGROUND: Eosinophils are prominent effectors of allergic inflammation. Taurine-chloramine (TauCl), a derivative of the amino acid taurine, shows antioxidant properties in different cell systems but its effects on eosinophils have not been reported. OBJECTIVE: To study the effects of TauCl and taurine on functional responses of isolated human eosinophils activated by different stimuli. METHODS: Human eosinophils were purified from the blood of healthy donors by a magnetic bead separation system. The effects of TauCl and taurine (0.1-1 mM) were investigated on the generation of superoxide anion (ferricytochrome-c reduction microassay), calcium signal (fluorimetry), p47phox-p67phox translocation (Western blot), leukotriene C4 (LTC4) production (enzymeimmunoassay), eosinophil peroxidase (EPO) release (spectrophotometry), eosinophil cationic protein (ECP) release (radioimmunoassay), apoptosis (flow cytometry with annexin V-propidium iodide), and nuclear factor-kappaB (NF-kappaB) activation (Western blot). RESULTS: TauCl inhibited superoxide anion generation triggered by N-formyl-Met-Leu-Phe (fMLP; 30 nM), phorbol myristate acetate (1 nM) and serum opsonized zymosan (0.5 mg/mL) with similar potency (IC50 approximately 200 microM) for the three stimuli, while taurine (0.1-1 mM) was scarcely effective. TauCl but not taurine inhibited p47phox-p67phox translocation. TauCl (200 microM) and taurine (1 mM) did not modify the [Ca2+]i responses to fMLP. TauCl inhibited the release of EPO (IC50 approximately 200 microM) and reduced ECP and LTC4 production from fMLP-activated eosinophils while taurine was without significant effects. TauCl (1 mM) did not change constitutive apoptosis but significantly attenuated the ability of granulocyte-monocyte colony-stimulating factor (GM-CSF) and IL-5 to prevent apoptosis. The activation of eosinophil NF-kappaB induced by GM-CSF and IL-5 was suppressed by TauCl. CONCLUSION: Taurine is without significant in vitro effects on human eosinophil functions but its derivative TauCl inhibits oxidative burst and generation of inflammatory mediators, and reverses the survival effect produced by inflammatory cytokines. Therefore, endogenous TauCl may help to suppress excessive inflammatory response in eosinophils at inflammatory sites.

Neutrophils as a novel source of eosinophil cationic protein in IgE-mediated processes.

The production of eosinophil cationic protein (ECP) in IgE-mediated diseases has been associated mainly with eosinophils, although no IgE-dependent ECP release has been observed in these cells. Because there is increasing evidence of neutrophil participation in allergic processes, we have examined whether human neutrophils from allergic patients were able to produce ECP by an IgE-dependent mechanism. After challenge with specific Ags to which the patients were sensitized, ECP release was detected in the culture medium. Furthermore, intracellular protein was detected by flow cytometry, immunofluorescence staining, and Western blotting. Expression at both mRNA and de novo protein synthesis were detected, respectively, by RT-PCR and radiolabeling with (35)S. Ag effect was mimicked by cell treatment with anti-IgE Abs or Abs against FcepsilonRI and galectin-3 (FcepsilonRI>galectin-3), but not against FcepsilonRII. These observations represent a novel view of neutrophils as possible source of ECP in IgE-dependent diseases.

Effects of TNFalpha on the human nasal mucosa in vivo.

BACKGROUND: TNFalpha is a cytokine that may contribute to the pathophysiology of airway inflammation. Inhalation of TNFalpha produces granulocyte recruitment and airway hyperresponsiveness in man. Anti-TNFalpha treatment may inhibit allergen-induced plasma exudation in guinea-pig airways. Increased nasal mucosal output of TNFalpha has been demonstrated in allergic rhinitis, but the effect of TNFalpha on the human nasal mucosa has not been examined in vivo. OBJECTIVE: To examine effects of topical TNFalpha on the human nasal mucosa in vivo. METHODS: In a dose-finding study, healthy subjects received intranasal TNFalpha (0-7.5 microg). Nasal lavages were carried out before as well as 10 min and 24 h post challenge and alpha(2)-macroglobulin was measured as an index of plasma exudation. In a second study, involving patients with allergic rhinitis examined out of season, a sham-controlled nasal challenge with TNFalpha (10 microg) was performed and followed 24 h later by an allergen challenge. Lavages were performed before the TNFalpha challenge, 24 h thereafter, and 10 min post allergen challenge. alpha(2)-Macroglobulin, eosinophil cationic protein (ECP), myeloperoxidase (MPO), and IL-8 were analyzed as indices of plasma exudation, eosinophil activity, neutrophil activity, and pro-inflammatory cytokine production, respectively. RESULTS: In the dose-finding study, TNFalpha produced significant increases in alpha(2)-macroglobulin 24h post challenge (p<0.01). In allergic rhinitis, 10 microg of TNFalpha also produced this effect (p<0.01) as well as increases in ECP and IL-8 (p<0.01). MPO was increased 24 h post challenge, but this change did not reach statistical significance. TNFalpha did not produce any acute effects and did not affect the responsiveness to allergen. CONCLUSION: The present study demonstrates that topical TNFalpha produces a human nasal inflammatory response. These data suggest a role of TNFalpha in nasal conditions characterized by mucosal inflammation.

Cis-9,trans-11-CLA exerts anti-inflammatory effects in human bronchial epithelial cells and eosinophils: comparison to trans-10,cis-12-CLA and to linoleic acid.

Interaction of eosinophils and bronchial epithelial cells plays a pivotal role in maintaining inflammatory airway disease. Since conjugated linoleic acids (CLA) are suggested to exert anti-inflammatory effects, one purpose of this study was to compare cis-9,trans-11-CLA and trans-10,cis-12-CLA with regard to their influence on the stimulus-induced activation of eosinophils. ECP (eosinophil cationic protein) released in co-culture of stimulated and CLA-treated eosinophils with stimulated bronchial epithelial cells (BEAS-2B) was measured and cis-9,trans-11-CLA was found to be most potent in inhibiting ECP formation. Further, expression of the activation markers CD69 and CD13 induced by various stimuli (TNF-alpha, IL-5, IL-3) was significantly reduced in the presence of cis-9,trans-11-CLA. Subsequently, various concentrations of cis-9,trans-11-CLA vs. linoleic acid (LA, cis-9,cis-12-octadecadienoic acid) were tested for the effect on proliferative response and release of the pro-inflammatory cytokine IL-8 in stimulated BEAS-2B. Addition of cis-9,trans-11-CLA attenuated cell growth and significantly reduced IL-8 production at mRNA and protein levels. In contrast, LA had a slight stimulating effect on proliferation and was less effective in reducing the cytokine release. It was demonstrated that the inhibitory effect of cis-9,trans-11-CLA on IL-8 production is mediated through activation of the nuclear receptor PPARgamma, since blocking the receptor with a selective antagonist (GW9662) restored the stimulus-induced enhancement in IL-8 mRNA expression and protein secretion. PPARgamma has previously been shown to be closely involved in the downregulation of inflammation during hyperresponsiveness related to pulmonary immune responses. Thus, targeting PPARgamma, cis-9,trans-11-CLA might be of therapeutic value in the focus of airway disease while ameliorating inflammatory processes by affecting epithelial and eosinophil functions.

Blockade of airway hyperresponsiveness and inflammation in a murine model of asthma by a prodrug of cysteine, L-2-oxothiazolidine-4-carboxylic acid.

Oxidative stress plays an important role in the pathogenesis of bronchial asthma. An excess production of reactive oxygen species (ROS) and defective endogenous antioxidant defense mechanisms may be present in asthma. Reduced glutathione (GSH) is one of the most important reducing agents against oxidant free radicals. A reducing agent, L-2-oxothiazolidine-4-carboxylic acid (OTC), a prodrug of cysteine, increases intracellular GSH. We have used a mouse model for asthma to determine effects of OTC on allergen-induced bronchial inflammation and airway hyper-responsiveness. The administration of OTC reduced bronchial inflammation and airway hyper-responsiveness. ROS generation in bronchoalveolar lavage fluids was increased by ovalbumin (OVA) inhalation, but this increase was diminished by administration of OTC. The increased IL-4, IL-5, IL-13, and eosinophil cationic protein levels in lungs after OVA inhalation were significantly reduced by the administration of OTC. In addition, the increased expression of ICAM-1, VCAM-1, RANTES, and eotaxin in lungs after OVA inhalation was significantly reduced by the administration of OTC. We also showed that the increased NF-kappaB levels in nuclear protein extracts of lung tissues at 72 h after OVA inhalation were decreased by the administration of OTC. These findings suggest that OTC may reduce airway inflammation and hyper-responsiveness through regulation of NF-kappaB activity.