Inflammation-related plasma and CSF biomarkers for multiple sclerosis.

Effective biomarkers for multiple sclerosis diagnosis, assessment of prognosis, and treatment responses, in particular those measurable in blood, are largely lacking. We have investigated a broad set of protein biomarkers in cerebrospinal fluid (CSF) and plasma using a highly sensitive proteomic immunoassay. Cases from two independent cohorts were compared with healthy controls and patients with other neurological diseases. We identified and replicated 10 cerebrospinal fluid proteins including IL-12B, CD5, MIP-1a, and CXCL9 which had a combined diagnostic efficacy similar to immunoglobulin G (IgG) index and neurofilament light chain (area under the curve [AUC] = 0.95). Two plasma proteins, OSM and HGF, were also associated with multiple sclerosis in comparison to healthy controls. Sensitivity and specificity of combined CSF and plasma markers for multiple sclerosis were 85.7% and 73.5%, respectively. In the discovery cohort, eotaxin-1 (CCL11) was associated with disease duration particularly in patients who had secondary progressive disease (PCSF < 4 × 10-5, Pplasma < 4 × 10-5), and plasma CCL20 was associated with disease severity (P = 4 × 10-5), although both require further validation. Treatment with natalizumab and fingolimod showed different compartmental changes in protein levels of CSF and peripheral blood, respectively, including many disease-associated markers (e.g., IL12B, CD5) showing potential application for both diagnosing disease and monitoring treatment efficacy. We report a number of multiple sclerosis biomarkers in CSF and plasma for early disease detection and potential indicators for disease activity. Of particular importance is the set of markers discovered in blood, where validated biomarkers are lacking.

Genetic Variation in Surfactant Protein-A2 Delays Resolution of Eosinophilia in Asthma.

Surfactant protein-A (SP-A) is an important mediator of pulmonary immunity. A specific genetic variation in SP-A2, corresponding to a glutamine (Q) to lysine (K) amino acid substitution at position 223 of the lectin domain, was shown to alter the ability of SP-A to inhibit eosinophil degranulation. Because a large subgroup of asthmatics have associated eosinophilia, often accompanied by inflammation associated with delayed clearance, our goal was to define how SP-A mediates eosinophil resolution in allergic airways and whether genetic variation affects this activity. Wild-type, SP-A knockout (SP-A KO) and humanized (SP-A2 223Q/Q, SP-A2 223K/K) C57BL/6 mice were challenged in an allergic OVA model, and parameters of inflammation were examined. Peripheral blood eosinophils were isolated to assess the effect of SP-A genetic variation on apoptosis and chemotaxis. Five days postchallenge, SP-A KO and humanized SP-A2 223K/K mice had persistent eosinophilia in bronchoalveolar lavage fluid compared with wild-type and SP-A2 223Q/Q mice, suggesting an impairment in eosinophil resolution. In vitro, human SP-A containing either the 223Q or the 223K allele was chemoattractant for eosinophils whereas only 223Q resulted in decreased eosinophil viability. Our results suggest that SP-A aids in the resolution of allergic airway inflammation by promoting eosinophil clearance from lung tissue through chemotaxis, independent of SP-A2 Q223K, and by inducing apoptosis of eosinophils, which is altered by the polymorphism.

Immunohistochemical differentiation of eosinophilic esophageal myositis from eosinophilic esophagitis.

BACKGROUND AND AIM: Eosinophilic esophagitis (EoE) is a Th2-mediated allergic disease of the esophageal epithelium, associated with antigen. We previously reported a case series for eosinophilic esophageal myositis (EoEM)-a novel eosinophilic gastrointestinal disorder defined as eosinophilic infiltration localized in the esophageal muscle layer-and diagnosed it by peroral endoscopic muscle biopsy. Here, we investigated the immunopathology of EoEM to differentiate it from EoE. METHODS: Histological analysis was performed for three cases of EoEM and EoE, respectively. The results were compared with those of two control samples (non-eosinophilic gastrointestinal disorder full-layer esophagus). Using immunofluorescence, we analyzed the expression of the chemokine receptor CCR3 and its ligands eotaxin-1 and eotaxin-3 to investigate the eosinophilic reaction. Additionally, we determined the expression patterns of desmoglein-1 in the esophageal epithelium, which shows dysregulated expression in EoE. RESULTS: Eosinophil infiltration was observed in the muscle layer (maximum number, 30, 36, 73/high-power field) and the epithelium (50, 44, 40/high-power field) for EoEM and EoE, respectively. In EoE esophageal epithelium, the number of eotaxin-3-positive epithelial cells was significantly increased together with CCR3-positive infiltrating cells. However, in EoEM, a number of eotaxin-1-positive and eotaxin-3-positive myocytes and vascular endothelial cells were increased in the esophageal muscle layer. A significant loss of desmoglein-1 expression was only observed in EoE, not in EoEM. CONCLUSIONS: Eotaxin-1 and eotaxin-3 expression on the smooth muscle and vessels plays a role in the pathogenesis of EoEM, while EoE shows an epithelial eotaxin-3-dominant immunoreaction. Thus, the EoEM immunological pattern displays clear differences from that of EoE.

IL-4 Modulates CCL11 and CCL20 Productions from IL-1ß-Stimulated Human Periodontal Ligament Cells.

BACKGROUND/AIMS: IL-4 is a multifunctional cytokine that is related with the pathological conditions of periodontal disease. However, it is uncertain whether IL-4 could control T cells migration in periodontal lesions. The aim of this study was to examine the effects of IL-4 on CCL11, which is a Th2-type chemokine, and CCL20, which is related with Th17 cells migration, productions from human periodontal ligament cells (HPDLCs). METHODS: CCL20 and CCL11 productions from HPDLCs were monitored by ELISA. Western blot analysis was performed to detect phosphorylations of signal transduction molecules in HPDLCs. RESULTS: IL-1ß could induce both CCL11 and CCL20 productions in HPDLCs. IL-4 enhanced CCL11 productions from IL-1ß-stimulated HPDLCs, though IL-4 inhibited CCL20 production. Western blot analysis showed that protein kinase B (Akt) and signal transducer and activator of transcription (STAT)6 pathways were highly activated in IL-4/IL-1ß-stimulated HPDLCs. Akt and STAT6 inhibitors decreased CCL11 production, but enhanced CCL20 production in HPDLCs stimulated with IL-4 and IL-1ß. CONCLUSIONS: These results mean that IL-4 enhanced Th2 cells migration in periodontal lesion to induce CCL11 production from HPDLCs. On the other hand, IL-4 inhibits Th17 cells accumulation in periodontally diseased tissues to inhibit CCL20 production. Therefore, IL-4 is positively related with the pathogenesis of periodontal disease to control chemokine productions in periodontal lesions.

Intraepithelial lymphocyte eotaxin-2 expression and perineural mast cell degranulation differentiate allergic/eosinophilic colitis from classic IBD.

OBJECTIVES: Allergic colitis shows overlap with classic inflammatory bowel disease (IBD). Clinically, allergic colitis is associated with dysmotility and abdominal pain, and mucosal eosinophilia is characteristic. We thus aimed to characterise mucosal changes in children with allergic colitis compared with normal tissue and classic IBD, focusing on potential interaction between eosinophils and mast cells with enteric neurones. METHODS: A total of 15 children with allergic colitis, 10 with Crohn disease (CD), 10 with ulcerative colitis (UC), and 10 histologically normal controls were studied. Mucosal biopsies were stained for CD3 T cells, Ki-67, eotaxin-1, and eotaxin-2. Eotaxin-2, IgE, and tryptase were localised compared with mucosal nerves, using neuronal markers neurofilament protein, neuron-specific enolase, and nerve growth factor receptor. RESULTS: Overall inflammation was greater in patients with CD and UC than in patients with allergic colitis. CD3 T-cell density was increased in patients with allergic colitis, similar to that in patients with CD but lower than in patients with UC, whereas eosinophil density was higher than in all other groups. Eotaxin-1 and -2 were localised to basolateral crypt epithelium in all specimens, with eotaxin-1+ lamina propria cells found in all of the colitis groups. Eotaxin-2+ intraepithelial lymphocyte (IEL) density was significantly higher in allergic colitis specimens than in all other groups. Mast cell degranulation was strikingly increased in patients with allergic colitis (12/15) compared with that in patients with UC (1/10) and CD (0/1). Tryptase and IgE colocalised on enteric neurons in patients with allergic colitis but rarely in patients with IBD. CONCLUSIONS: Eotaxin-2+ IELs may contribute to the periepithelial eosinophil accumulation characteristic of allergic colitis. The colocalisation of IgE and tryptase with mucosal enteric nerves is likely to promote the dysmotility and visceral hyperalgesia classically seen in allergic gastrointestinal inflammation.

CCL11 as a potential diagnostic marker for asthma?

OBJECTIVE: Asthma is an inflammatory airway disease characterized by airway eosinophilia, in which CCL11 (eotaxin) plays a crucial role. The aim of study is to determine the elevation of CCL11 levels in bronchoalveolar lavage fluid (BALF), blood, exhaled breath condensate (EBC) and sputum in asthma patients and to identify which medium yields the most significant change in CCL11 level. METHODS: The databases of PubMed, Embase and Cochrane Centre Register of Controlled Trials were systematically searched from inception to September 2013. Controlled clinical trials that focused on CCL11 concentrations in asthma patients and controls, and their correlations with other asthma indicators were obtained. Data were analysed using Stata 12.0. RESULTS: Thirty studies were included in this investigation. CCL11 levels in blood, EBC and sputum were significantly higher in asthma patients than in healthy subjects. Sputum CCL11 concentrations were significantly elevated in unstable asthma patients versus stable asthma patients and in uncontrolled asthma patients versus partially controlled asthma patients. CCL11 levels in sputum and blood were negatively correlated with the lung function as measured by FEV1% predicted, and were positively correlated with BALF, EBC and sputum eosinophil counts. Similarly, CCL11 concentrations were positively correlated with eosinophil cationic protein in EBC, blood and sputum as well as with interleukin-5 in sputum and fractional exhaled nitric oxide in EBC. Steroid treatment had no significant effect on CCL11 levels. CONCLUSIONS: CCL11 is a potentially useful biomarker for the diagnosis and assessment of asthma severity and control, especially in sputum. CCL11 is crucial in eosinophil chemoattraction and activation in asthma pathogenesis. Further studies using anti-CCL11 approaches are needed to confirm a role for CCL11 in asthma pathogenesis particularly in patients with more severe disease.

Prognostic impact of cytokines and chemokines in bronchoalveolar lavage fluid on acute exacerbation of fibrosing interstitial lung disease.

BACKGROUND AND OBJECTIVE: Acute exacerbation of fibrosing interstitial lung disease (AE-FILD) is a serious condition with a high mortality rate. We aimed to comprehensively analyze cytokines in bronchoalveolar lavage fluid and their association with the clinical course of AE-FILD. METHODS: We retrospectively enrolled 60 patients with AE-FILD who underwent bronchoalveolar lavage. We comprehensively measured 44 cytokines and chemokines in the obtained bronchoalveolar lavage fluid using a Luminex analyzer. Patients were grouped into those who died within 90 days (non-survival group) and survived beyond 90 days (survival group) to investigate the association of the levels of cytokines and chemokines with mortality. RESULTS: The levels of matrix metalloproteinase 1 (p = 0.003), granulocyte-macrophage colony-stimulating factor (p = 0.040), interleukin 6 (p = 0.047), interleukin 8 (p = 0.050), monocyte chemoattractant protein-1 (p = 0.043), and eotaxin (p = 0.044) were significantly higher in the non-survival group than in the survival group. In the receiver operating characteristic analysis, their areas under the curve were 0.80, 0.68, 0.71, 0.70, 0.70, and 0.72, respectively. Using machine learning with these six cytokines and chemokines, the predictive accuracy for the survival group was 0.94. CONCLUSIONS: Our study demonstrated that several cytokines and chemokines in bronchoalveolar lavage fluid could be prognostic predictors in patients with AE-FILD.

Sputum mediator profiling and relationship to airway wall geometry imaging in severe asthma.

BACKGROUND: Severe asthma is a heterogeneous disease and the relationship between airway inflammation and airway remodelling is poorly understood. We sought to define sputum mediator profiles in severe asthmatics categorised by CT-determined airway geometry and sputum differential cell counts. METHODS: In a single centre cross-sectional observational study we recruited 59 subjects with severe asthma that underwent sputum induction and thoracic CT. Quantitative CT analysis of the apical segment of the right upper lobe (RB1) was performed. Forty-one mediators in sputum samples were measured of which 21 mediators that were assessable in >50% of samples were included in the analyses. RESULTS: Independent of airway geometry, sputum MMP9 and IL-1ß were elevated in those groups with a high sputum neutrophil count while sputum ICAM was elevated in those subjects with a low sputum neutrophil count. In contrast, sputum CCL11, IL-1α and fibrinogen were different in groups stratified by both sputum neutrophil count and airway geometry. Sputum CCL11 concentration was elevated in subjects with a low sputum neutrophil count and high luminal and total RB1 area, whereas sputum IL1α was increased in subjects with a high sputum neutrophil count and low total RB1 area. Sputum fibrinogen was elevated in those subjects with RB1 luminal narrowing and in those subjects with neutrophilic inflammation without luminal narrowing. CONCLUSIONS: We have demonstrated that sputum mediator profiling reveals a number of associations with airway geometry. Whether these findings reflect important biological phenotypes that might inform stratified medicine approaches requires further investigation.

[Effects of andrographolide on the expression of eosinophil granulocytes and possible mechanisms].

OBJECTIVE: Andrographolide, the active component in andrographis paniculata, has potent anti-inflammatory actions. This study aimed to evaluate the effects of andrographolide on eosinophil granulocytes (EOS) and the expression of eotaxin and IL-5 in mice with asthma. METHODS: BALB/c mice were randomly assigned into normal control, asthma, budesonide treatment and andrographolide treatment groups (n=8 each). Mice in the latter three groups were sensitized and challenged with ovalbumin (OVA) to induce asthma. ELISA was used to detect the concentrations of eotaxin and IL-5 in bronchoalveolar lavage fluid (BALF) and peripheral blood. The expression of eotaxin mRNA and IL-5 mRNA in lung tissues was detected by real-time quantitative PCR. RESULTS: Andrographolide treatment significantly decreased EOS count in BALF (P<0.05) and the effect of andrographolide was better than the effect of budesonide. Andrographolide treatment significantly down-regulated the expression of eotaxin and IL-5 in BALF, lung eotaxin mRNA expression and blood IL-5 expression (P<0.05), but the effects of andrographolide were poorer than the effects of budesonide. Andrographolide treatment resulted in a decrease in blood eotaxin expression and lung IL-5 mRNA expression and the effects of andrographolide were similar to budesonide. CONCLUSIONS: Andrographolide can down-regulate the expression of IL-5 and eotaxin and thus suppress the inflitration of EOS in a mouse model of asthma.

Immune profile and Epstein-Barr virus infection in acute interstitial nephritis: an immunohistochemical study in 78 patients.

BACKGROUND: Acute interstitial nephritis (AIN) is a common cause of acute kidney injury and is characterised by a dense interstitial cellular infiltrate, which has not been well defined. Previous studies have demonstrated a correlation between Epstein-Barr virus (EBV) infection and AIN. The purpose of our study was to define the nature of the interstitial immune infiltrate and to investigate the possibility of renal infection with EBV. METHODS: Seventy-eight patients with AIN were identified from renal biopsy reports in a single centre over an 18-year period. Immunohistochemical staining was performed to define the cellular infiltrate. In situ hybridization and immunohistology were used to detect EBV. RESULTS: A positive correlation between CD68 macrophage infiltration and serum creatinine concentration at presentation was identified. IL-4, eotaxin, CCR3, CCR5 and VCAM-1 were all expressed in biopsies of AIN. Using in situ hybridization and immunohistochemistry, EBV was not detected in any of the AIN sections analysed. CONCLUSION: This study has assessed the nature of the interstitial infiltrate in AIN. EBV was not detected in the renal biopsies, suggesting that EBV is not a pathogenetic factor in AIN.

Clara cell 10-kD protein suppresses chitinase 3-like 1 expression associated with eosinophilic chronic rhinosinusitis.

RATIONALE: Clara cell 10-kD (CC10) protein, an antiinflammatory molecule, is involved in inflammatory upper airway diseases, but its regulatory role is unclear, particularly in the process of chronic rhinosinusitis (CRS). OBJECTIVES: To investigate the regulatory mechanisms of CC10 in eosinophilic CRS (ECRS) using an allergic mouse model. METHODS: Homozygous CC10-knockout mice were used to establish an allergic ECRS model. Phenotypic changes were examined by histology, cytokine ELISA, and gene microarray analysis. Differential expression of chitinase 3-like 1 (CHI3L1) was verified by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. The functional role of CHI3L1 in vivo was assessed by the use of anti-CHI3L1 antibody in ECRS mice. CHI3L1 gene expression regulated by inflammatory cytokines and CC10 protein was performed using BEAS-2B cell line. MEASUREMENTS AND MAIN RESULTS: Compared with wild-type mice, a significantly greater extent of inflammatory cell infiltration and tissue remodeling was found in CC10-knockout ECRS mice, which was associated with significantly higher levels of various cytokines and eotaxin-1. CHI3L1 was up-regulated in ECRS mice with a significant further increase in CC10-knockout mice. Anti-CHI3L1 treatment markedly ameliorated eosinophilic inflammation. Furthermore, nasal mucosal CC10 gene transfer in CC10-knockout mice attenuated eosinophilic inflammation and suppressed the levels of CHI3L1. Moreover, significantly up-regulated expression of CHI3L1 was noted in human ECRS. IL-1beta, tumor necrosis factor-alpha, and IL-13 were found to up-regulate CHI3L1 expression in BEAS-2B cells, whereas CC10 inhibited such up-regulation. CONCLUSIONS: These results suggest that CHI3L1 is a novel molecule involved in ECRS and that CC10 plays a regulatory role in ECRS, presumably by attenuating CHI3L1 expression.

Activation of innate immunity accelerates sialoadenitis in a mouse model for Sjögren's syndrome-like disease.

OBJECTIVE: Sjögren's syndrome is a chronic autoimmune disorder characterized by progressive lymphocytic infiltration within the salivary and lacrimal glands. This study was undertaken to investigate the effects of innate immunity activation on sialoadenitis in a mouse strain genetically susceptible for development of SS-like disease. METHODS: Female New Zealand Black X New Zealand White F1 mice were repeatedly treated with toll-like 3 receptor agonist poly(I:C). Submandibular glands were investigated at different time points for sialoadenitis by immunohistochemistry and for gene expression of different chemokines by quantitative PCR. Submandibular gland-infiltrating cells were characterized by flow cytometry. RESULTS: Poly(I:C) treatment significantly upregulated the expression of multiple chemokines within the submandibular glands. The severity and incidence of sialoadenitis was considerably higher in poly(I:C)-treated mice. There was a preponderance of dendritic cells and NK cells in the initial inflammatory cell infiltrates, and these were followed by CD4+ T cells. CONCLUSIONS: Our data clearly demonstrate that systemic activation of innate immunity accelerates sialoadenitis in a mouse model for SS-like disease. These findings suggest that chronic activation of innate immunity can influence certain features of SS.

Persistence of increased Eotaxin-1 (CCL11) level in tears of patients wearing contact lenses: a long-term follow-up study.

BACKGROUND: Eotaxin-1 (CCL11) is a potent eosinophil chemotactic and activating peptide that may be implicated in the pathogenesis of chronic allergic eye disease and has been associated with the wearing of contact lenses (CL) in patients with contact lens papillary conjunctivitis (CLPC). The purpose of this study was to study eotaxin-1 expression in the tears of long-term CL wearers. PATIENTS AND METHODS: Tears were collected with glass capillaries from 15 patients (2 male, 13 female) with various degree of CLPC at 2-year intervals. CLPC severity was graded from 0 to 4 with reference to standard slit-lamp photographs of the superior tarsal conjunctiva. The eotaxin-1 level in the tears was measured by an ELISA, using mouse anti-human eotaxin monoclonal antibodies. RESULTS: The mean age was 32.5 ± 13.3 years (range: 17 - 69 years). The mean interval between the tear collections was 30 ± 4.8 months. The mean concentration of eotaxin was 2150 ± 477 pg/mL and 2486 ± 810 pg/mL for the first and second series, respectively. The difference was not statistically significant (paired Wilcoxon/Kruskal-Wallis, p = 0.803). The mean score of papilla grade was 1.26 ± 0.18 for the first sample and 1.40 ± 0.19 two years later. There was no significant difference of grading between the two time periods (paired Wilcoxon/Kruskal-Wallis, p = 0.751). CONCLUSIONS: the eotaxin-1 level remains up-regulated over a long time period in patients wearing CL, most of them with chronic CLPC. Eotaxin may play a role in the pathogenesis of contact lens intolerance.

Salbutamol combined with budesonide in treatment of pediatric bronchial asthma and its effect on eosinophils.

BACKGROUND: The aim of this study was to investigate and discuss the salbutamol combined with budesonide in treatment of pediatric bronchial asthma (BA) and its effect on eosinophils (EOS). METHODS: Ninety-eight BA children admitted and treated in our hospital from July 2016 to June 2017 were collected and divided into control group (N.=49) and observation group (N.=49) according to random number table. The children in control group were treated with budesonide and those in observation group were treated with salbutamol combined with budesonide. The clinical efficacy, pulmonary functions and levels of T-lymphocyte subsets (including cluster of differentiation 3 (CD3)+, CD4+, CD8+ and CD4+/CD8+) in the immune system between two groups were compared after the treatment; the levels of eosinophil cationic protein (ECP) and eotaxin in the children were compared before the treatment and at 1, 4 and 8 weeks after the treatment; the changes in EOS counts in blood and induced sputum of the children before and after the treatment were compared, and the EOS apoptosis rate was compared at 1, 4 and 8 weeks after the treatment. RESULTS: The effective rate of treatment in observation group was significantly higher than that in control group (P<0.05). After the treatment, the indexes of pulmonary function in observation group were obviously better than those in control group (P<0.05). Compared with those in control group, the levels of CD3+, CD4+ and CD4+/CD8+ of the children in observation group were elevated remarkably, while the CD8+ level was lowered (P<0.05). The levels of ECP and eotaxin in the two groups were decreased after the treatment compared with those before the treatment, and the levels in observation group were superior to those in control group (P<0.05). After the treatment, the EOS counts of both groups of children were lower than those before the treatment, and the decrease in observation group was more notable than that in control group. At 1, 4 and 8 weeks after the treatment, the EOS apoptosis rate in observation group was obviously higher than that in control group (P<0.05). CONCLUSIONS: The treatment of salbutamol combined with budesonide for pediatric BA has significant therapeutic effects; it can restore the pulmonary functions rapidly and improve the immunity of the lung, reduce the levels of eotaxin, ECP and EOS of the child patients and promote EOS apoptosis.

Detection of respiratory viruses and the associated chemokine responses in serious acute respiratory illness.

BACKGROUND: A specific diagnosis of a lower respiratory viral infection is often difficult despite frequent clinical suspicion. This low diagnostic yield may be improved by use of sensitive detection methods and biomarkers. METHODS: The prevalence, clinical predictors and inflammatory mediator profile of respiratory viral infection in serious acute respiratory illness were investigated. Sequential bronchoalveolar lavage (BAL) fluids from all patients hospitalised with acute respiratory illness over 12 months (n=283) were tested for the presence of 17 respiratory viruses by multiplex PCR assay and for newly discovered respiratory viruses (bocavirus, WU and KI polyomaviruses) by single-target PCR. BAL samples also underwent conventional testing (direct immunoflorescence and viral culture) for respiratory virus at the clinician's discretion. 27 inflammatory mediators were measured in a subset of the patients (n=64) using a multiplex immunoassay. RESULTS: 39 respiratory viruses were detected in 37 (13.1% of total) patients by molecular testing, including rhinovirus (n=13), influenza virus (n=8), respiratory syncytial virus (n=6), human metapneumovirus (n=3), coronavirus NL63 (n=2), parainfluenza virus (n=2), adenovirus (n=1) and newly discovered viruses (n=4). Molecular methods were 3.8-fold more sensitive than conventional methods. Clinical characteristics alone were insufficient to separate patients with and without respiratory virus. The presence of respiratory virus was associated with increased levels of interferon gamma-inducible protein 10 (IP-10) (p<0.001) and eotaxin-1 (p=0.017) in BAL. CONCLUSIONS: Respiratory viruses can be found in patients with serious acute respiratory illness by use of PCR assays more frequently than previously appreciated. IP-10 may be a useful biomarker for respiratory viral infection.

Eotaxin-1 in exhaled breath condensate of stable and unstable asthma patients.

BACKGROUND: Airway eosinophilia is considered a central event in the pathogenesis of asthma. Eotaxin plays a key role in selective eosinophil accumulation in the airways and, subsequently, their activation and degranulation. The study was undertaken to evaluate eotaxin-1 levels in the exhaled breath condensate (EBC) of asthmatics with different degrees of asthma severity and to establish the possible correlation of these measurements with other recognized parameters of airway inflammation. METHODS: EBC was collected from 46 patients with allergic asthma (14 with steroid-naïve asthma, 16 with ICS-treated, stable asthma, 16 with ICS-treated unstable asthma) and 12 healthy volunteers. Concentrations of eotaxin-1 were measured by ELISA. RESULTS: In the three groups of asthmatics, eotaxin-1 concentrations in EBC were significantly higher compared with healthy volunteers (steroid-naïve asthma: 9.70 pg/ml +/- 1.70, stable ICS-treated asthma: 10.45 +/- 2.00, unstable ICS-treated asthma: 17.97 +/- 3.60, healthy volunteers: 6.24 +/- 0.70). Eotaxin-1 levels were significantly higher in patients with unstable asthma than in the two groups with stable disease. We observed statistically significant correlations between the concentrations of eotaxin-1 in EBC and exhaled nitric oxide (F(ENO)) or serum eosinophil cationic protein (ECP) in the three studied groups of asthmatics. We also discovered a significantly positive correlation between eotaxin-1 in EBC and blood eosinophil count in the groups of patients with unstable asthma and steroid-naïve asthma. CONCLUSIONS: Measurements of eotaxin-1 in the EBC of asthma patients may provide another useful diagnostic tool for detecting and monitoring airway inflammation and disease severity.

Suppressive effects of formoterol and salmeterol on eotaxin-1 in bronchial epithelial cells.

Eotaxin-1 (CCL11), an eosinophil-specific C-C chemokine, is a potent chemoattractant for mobilization of eosinophils into airways after allergic stimulation. Eotaxin-1 recruits eosinophils into inflammatory sites, and may play a role in the pathogenesis of asthma. Formoterol and salmeterol are two inhaled long acting beta(2) adrenoceptor agonists (LABAs), widely used for the local treatment of asthma. However, little is known about their effects on the eotaxin-1 expression of bronchial epithelial cells. BEAS-2B cells were stimulated by adding IL-4 with or without 2 h pre-treatment of formoterol or salmeterol. The protein and mRNA expression of eotaxin-1 were measured by ELISA assay and real-time PCR, respectively. Effects of formoterol and salmeterol on nuclear and cytosolic pSTAT-6 expression were evaluated by Western blot and immunofluorescence study. Formoterol and salmeterol (10(-7)-10(-10) m) significantly down-regulated IL-4- induced eotaxin-1 expression in BEAS-2B cells. A specific beta(2) adrenoceptor antagonist (ICI 118,551) reversed their suppression of eotaxin-1 production. Forskolin, an cAMP activator, could also suppress the expression of eotaxin-1 by IL-4 in a dose dependent manner (10(-7)-10(-10 )m). The western blot and immunofluorescence studies demonstrated that formoterol 10(-7 )m suppressed the nuclear expression of pSTAT-6. Formoterol and salmeterol, two inhaled long-acting beta(2) agonists, down-regulated IL-4- induced eotaxin-1 expression in BEAS-2B cells. The effect was mediated via the beta(2) adrenoceptor, and cAMP. Formoterol significantly down-regulated pSTAT6 at higher concentration, and further turned off the IL-4 signaling pathway.

Smoking affects eotaxin levels in asthma patients.

BACKGROUND: Chronic airway inflammation is most important pathological finding in asthma. Cigarette smoking may modify type of inflammation as well as may influence disease severity and response to the treatment. OBJECTIVE: Thus the aim of this study was to investigate whether cigarette smoking may have an influence on the levels of eotaxin-1, eotaxin-2, eotaxin-3 and IL-5 in patients with stable mild/moderate asthma. METHODS: 45 steroid naive asthmatics (mean age: 55.2 +/- 2.2 yrs) and 23 "healthy" smokers and non-smokers control subjects (mean age: 54.4 +/- 9.7 yrs) were investigated. Asthmatics were divided into two subgroups according to their smoking histories: asthmatic smokers (n = 19) who currently smoke and have a history of > 10 pack-years and asthmatic never-smokers (n = 26). BAL and induced sputum were performed. Cytospins of induced sputum and BAL were stained with May-Grunwald-Giemsa for differential cell counts. Eotaxin-1, eotaxin-2, eotaxin-3 and IL-5 concentrations in serum, sputum and BAL supernatant was measured using a commercial ELISA kit. RESULTS: In sputum supernatant from asthma smokers was significantly higher concentration of eotaxin-1 than in non-smokers asthmatics (203.4 +/- 10.0 vs. 140.2 +/- 9.5 respectively, p < 0.05). In non-smokers asthma patients levels of BAL eotaxin-1 strongly related to percent and absolute numbers of BAL eosinophils and neutrophils (Rs = 0.737 and Rs = 0.514 respectively, p < 0.05). The number and percent of sputum neutrophils and eosinophils, obtained from smokers asthmatics, significantly correlated with eotaxin-2 concentration in sputum supernatant (Rs = 0.58 and Rs = 0.75 respectively, p < 0.05). IL-5 levels in the serum and sputum from asthmatic never-smokers were significantly higher than they were from asthmatic smokers and "healthy" smokers. Asthmatic never-smokers showed a significantly higher amount of IL-5 in serum and sputum than the asthmatic smokers showed. CONCLUSIONS: This study showed the elevated levels of sputum eotaxin-1 as well as serum, sputum and BAL eotaxin-2 in asthmatic smokers without a significant increase of eosinophils compared to asthmatic never-smokers. The eotaxin concentrations were related not only with number of eosinophils but also with the number of neutrophils in all the studied tissue compartments. The data herein permits a suggestion that smoking may influence change in asthmatic airway inflammation by stimulating the production of eotaxins.

Epidermal expression of eotaxins and thymic stromal lymphopoietin in eosinophil rich dermatoses.

Eosinophils are seen in a number of dermatologic conditions. While the extent of their function in these diseases remains to be fully elucidated, pathogenic activity in bullous pemphigoid suggests a more significant role than previously thought. Several dermatoses have a fairly characteristic histologic morphology of eosinophil infiltration. We hypothesized that epidermal expression of eotaxins and TSLP would differ by disease, perhaps explaining the different histologic morphologies. We performed a retrospective study of eosinophil rich dermatoses to perform immunohistochemistry. We collected 49 specimens composed of bullous pemphigoid (n = 15), atopic dermatitis (n = 12), drug rash (n = 8), arthropod assault (n = 5), and non-bullous pemphigoid eosinophilic spongiosis (n = 5). We used lichen planus (n = 4) as a control for lymphocyte-mediated inflammation. TSLP was diffusely expressed in all epidermal samples, whereas eotaxins demonstrated a weaker staining. Eotaxins and TSLP demonstrated a gradient between basal and spinous keratinocytes. The correlation between overall basal keratinocyte and spinous keratinocyte staining of eotaxins and TSLP with the number of eosinophils demonstrated a significant correlation between eotaxin-1 (R = 0.404, P = 0.004), eotaxin-2 (R = 0.576, P < 0.001), and eotaxin-3 (R = 0.512, P < 0.001), but not TSLP (R = 0.164, P = 0.251). These remained significant after correcting for multiple comparisons. While we were unable to detect significant differences in epidermal expression of eotaxins and TSLP in various eosinophil rich dermatoses, we identified a significant correlation of spinous keratinocyte eotaxin staining with tissue eosinophilia. Our identification of a correlation of spinous keratinocyte eotaxin staining with tissue eosinophilia may provide insight into local eosinophil chemotaxis.

Small interfering RNA against interleukin-5 decreases airway eosinophilia and hyper-responsiveness.

Interleukin-5 (IL-5) has been suggested to be involved in the development of airway hyper-responsiveness (AHR). Both clinical and experimental investigations have shown strong correlation between the presence of eosinophils and AHR. In this study, we used small interfering RNA (siRNA) as an approach to inhibiting the expression of IL-5 and reducing AHR. siRNAs targeting IL-5 were characterized in vitro, and siRNA-expressing lentiviruses were administered intratracheally to OVA-sensitized BALB/c mice. AHR, cytokine levels, serum levels of OVA-specific antibodies and infiltration of inflammatory cells were analyzed to investigate the effects of siRNA in an OVA-induced murine model of asthma. Lentivirus-delivered siRNA targeting IL-5 efficiently moderated the characteristics of asthma, including AHR, cellular infiltration of lung tissues, eotaxin levels in the bronchoalveolar lavage fluid and IL-5 mRNA levels in lungs in the mouse model of asthma. However, there was no effect on OVA-specific IgE level. These data demonstrate that siRNA delivered by the lentiviral system is an efficacious therapeutic strategy for asthma.