Anti-inflammatory deficiencies in neutrophilic asthma: reduced galectin-3 and IL-1RA/IL-1β

Background

Galectin-3 (gal-3), a member of the β-galactoside-binding animal lectins, is involved in the recruitment, activation and removal of neutrophils. Neutrophilic asthma is characterized by a persistent elevation of airway neutrophils and impaired efferocytosis. We hypothesized that sputum gal-3 would be reduced in neutrophilic asthma and the expression of gal-3 would be associated with other markers of neutrophilic inflammation.

Methods

Adults with asthma (n = 80) underwent a sputum induction following clinical assessment and blood collection. Sputum was dispersed for a differential cell count and ELISA assessment of gal-3, gal-3 binding protein (BP), interleukin (IL)-1β, IL-1 receptor antagonist (RA), IL-8 and IL-6. Gal-3 and gal-3BP immunoreactivity were assessed in mixed sputum cells.

Results

Sputum gal-3 (median, (q1,q3)) was significantly reduced in neutrophilic asthma (183 ng/mL (91,287)) compared with eosinophilic (293 ng/mL (188,471), p = 0.021) and paucigranulocytic asthma (399 ng/mL (213,514), p = 0.004). The gal-3/gal-3BP ratio and IL-1RA/IL-1β ratio were significantly reduced, while gal-3BP and IL-1β were significantly elevated in neutrophilic asthma compared with eosinophilic and paucigranulocytic asthma.

Conclusion

Patients with neutrophilic asthma have impairment in anti-inflammatory ratio of gal-3/gal-3BP and IL-1RA/IL-1β which provides a further framework for exploration into pathologic mechanisms of asthma phenotypes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0163-5) contains supplementary material, which is available to authorized users.     

High mobility group protein B1 (HMGB1) in Asthma: comparison of patients with chronic obstructive pulmonary disease and healthy controls

High mobility group protein B1 (HMGB1) has been implicated as an important mediator in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). However, the expression of HMGB1 in plasma and sputum of patients with asthma and COPD across disease severity needs to be defined. The objective of the study was to examine the induced sputum and plasma concentrations of HMGB1 in COPD and asthmatic patients to determine differences in HMGB1 levels between these diseases and their relationship with airway obstruction and inflammatory patterns. A total of 147 participants were enrolled in this study. The participants included 34 control subjects, 61 patients with persistent asthma (according to the Global Initiative for Asthma [GINA] guidelines) and 47 patients with stable COPD (stratified by Global Initiative for Chronic Obstructive Lung Disease [GOLD] status). Spirometry was performed before sputum induction. HMGB1 levels in induced sputum and plasma were determined by enzyme-linked immunosorbent assay. Sputum and plasma concentrations of HMGB1 in patients with asthma and COPD were significantly higher than concentrations in control subjects and were significantly negatively correlated with forced expiratory volume in 1 s (FEV(1)), FEV(1) (% predicted) in all 147 participants. The levels of HMGB1 in induced sputum of COPD patients were significantly higher than those of asthma patients and healthy controls (P < 0.001). This difference was present even after adjusting for sex, age, smoking status, daily dose of inhaled corticosteroids and disease severity. There were no significant differences in HMGB1 levels between patients with eosinophilic and noneosinophilic asthma. HMGB1 levels in asthmatic and COPD patients were positively correlated with neutrophil counts and percentage of neutrophils. In multivariate analysis, the two diseases (asthma and COPD) and disease severity were independent predictors of sputum HMGB1, but not smoking, age or use of inhaled corticosteroids. In conclusion, these data support a potential role for HMGB1 as a biomarker and diagnostic tool for the differential diagnosis of asthma and COPD. The importance of this observation on asthma and COPD mechanisms and outcomes should be further investigated in large prospective studies.     

Ozone-induced inflammation assessed in sputum and bronchial lavage fluid from asthmatics: a new noninvasive tool in epidemiologic studies on air pollution and asthma

We investigated correlations between ozone-induced increases in inflammatory markers in induced sputum and in bronchial lavage fluid. Sixteen volunteers with intermittent asthma participated in a placebo-controlled parallel study with two exposures. Six days before and 16 h after the first exposure to ozone (0.4 ppm during 2 h) sputum was induced with hypertonic saline. This resulted in a significant increase in the sputum levels of eosinophil cationic protein (ECP; 1.8-fold; p = .03), neutrophil elastase (5.0-fold; p = .005) and the total cell number (1.6-fold; p = .02). After 4 weeks, a second exposure was randomized for air or ozone. Six days before and 16 h after the second exposure a bronchial lavage was performed. ECP values in sputum and in bronchial lavage fluid obtained after ozone correlated significantly (Rs = .79; p = .04), as did interleukin-8 (IL-8) values (Rs = .86; p = .01), and the percentage eosinophils (Rs = .89; p = .007). Moreover, the ozone-induced changes in percentage eosinophils observed in sputum and lavage fluid were highly correlated (Rs = .93; p = .003). In conclusion, changes in eosinophils, IL-8, and ECP markers induced by ozone and measured in sputum reflect the inflammatory responses in the lower airways of asthmatics, and may provide a noninvasive tool in epidemiologic studies on air pollution and asthma.     

Effects after inhalation of (1-->3)-beta-D-glucan in healthy humans

BACKGROUND AND AIM: This study was performed to assess the effects of an exposure to a pure (1-->3)-beta-D-glucan, a cell wall component of fungi, plants and certain bacteria. METHODS: Twenty-one healthy subjects inhaled saline or (1-->3)-beta-D-glucan suspended in saline in a random, double-blind, cross-over design. They were examined before exposure and 24 and 72h afterwards with spirometry, blood sampling and collection of induced sputum. Differential cell counts and eosinophilic cationic protein (ECP) were determined in blood and sputum, and myeloperoxidase (MPO), tumour necrosis factor-alpha (TNF-alpha), and interleukin (IL)-8 and IL-10 were determined in sputum supernatants. TNF-alpha was determined after cultivation of blood mononuclear cells. RESULTS: In sputum, inhalation of saline caused a significant increase in ECP and TNF-alpha. (1-->3)-beta-D-Glucan inhalation caused a further increase in these cytokines, although not statistically significantly different from the increase induced by inhalation of saline alone. In blood, the number of eosinophils was significantly decreased 72 h after the challenge with (1-->3)-beta-D-glucan. This effect was not found after the inhalation of saline alone. TNF-alpha production from stimulated blood mononuclear cells was significantly decreased 72 h after the (1-->3)-beta-D-glucan inhalation as compared with the increase induced by saline inhalation. CONCLUSIONS: The results suggest that (1-->3)-beta-D-glucan causes a different type of response as compared with inflammatory agents such as bacterial endotoxin that cause a neutrophil-dominated inflammatory response.     

Bronchial macrophages in asthmatics reveal decreased CD16 expression and substantial levels of receptors for IL-10, but not IL-4 and IL-7

The role of different subpopulations of bronchial macrophages (BMs) in asthma pathogenesis has not yet been completely elucidated. In addition, little is known about potential in vivo responsiveness of BMs to pro- and anti-inflam-matory cytokines present in the bronchial milieu. We aimed to characterize asthmatic patients' BM subpopulations delineated by common markers of macrophage/monocyte cells, CD16 and CD14, and subsequently to analyze cytokine receptor expression on those subsets. Subjects included eighteen patients with moderate asthma (six steroid-naive and twelve steroid-treated) and ten healthy control subjects. Flow cytometry was used to analyze phenotypical features of BMs including expression of receptors for IL-10, IL-4 and IL-7. Exhaled nitric oxide analysis and induced sputum eosinophil counts were used to assess airway inflammation. BMs from both steroid-naive and steroid-treated asthmatic patients showed significantly decreased expression of CD16, as compared to healthy subjects' BMs. CD16, but not CD14, expression inversely correlated with exhaled nitric oxide levels and sputum eosinophilia. Short-term administration of inhaled cortiocosteroids (ICS) in steroid-naive asthmatic patients led to significant reduction of CD16 expression and enhancement of CD14 expression. Next, we analyzed the expression of receptors for IL-10, IL-4 and IL-7 on the surface of BM subpopulations characterized by different levels of CD14 and CD16 expression. We observed substantial levels of IL-10R on the surface of BMs collected from asthmatic and healthy subjects. Interestingly, IL-10R was found mostly on those macrophages that co-expressed CD14. In contrast, independently on co-expression of CD14, the levels of IL-4R and IL-7R on BMs were low in both asthmatic and healthy subjects. The results suggest that different BM subsets may be differentially involved in regulating the inflammatory response in allergic asthma.     

Galectin-10, a potential biomarker of eosinophilic airway inflammation

Measurement of eosinophilic airway inflammation can assist in the diagnosis of allergic asthma and in the management of exacerbations, however its clinical implementation remains difficult. Galectin-10 has been associated with eosinophilic inflammation and has the potential to be used as a surrogate biomarker. This study aimed to assess the relationship between galectin-10 in sputum with sputum eosinophil counts, the current gold standard of eosinophil inflammation in the lung. Thirty-eight sputum samples were processed for both eosinophil counts by cytospins and semi-quantitative measurements of galectin-10 by western blots. A strong association was observed between galectin-10 levels in sputum and sputum eosinophil measurements, and they accurately determined sputum eosinophilia. The results support the potential for galectin-10 to be used as a surrogate biomarker of eosinophilic airway inflammation.     

Increase in markers of airway inflammation after ozone exposure can be observed also in stable treated asthmatics with minimal functional response to ozone

Background

The discrepancy between functional and inflammatory airway response to ozone has been reported in normal subjects, but few data are available for stable asthmatics regularly treated with inhaled corticosteroids.

Methods

Twenty-three well controlled, regularly treated, mild-to-moderate asthmatic patients underwent two sequential randomised exposures to either filtered air or ozone (0.3 ppm for 2 hours) in a challenge chamber. Pulmonary function (PF) was monitored, and patients with FEV1 decrease greater than 10% from pre-challenge value were considered as responders. Immediately after each exposure, exhaled breath condensate (EBC) was collected to measure malondialdehyde (MDA). Six hours after each exposure, PF and EBC collection were repeated, and sputum was induced to measure inflammatory cell counts and soluble mediators (IL-8 and neutrophil elastase). The response to ozone was also evaluated according to the presence of polymorphism in oxidative stress related NQO1 and GSTM1 genes.

Results

After ozone exposure, sputum neutrophils significantly increased in responders (n = 8), but not in nonresponders (n = 15). Other markers of neutrophil activation in sputum supernatant and MDA in EBC significantly increased in all patients, but only in nonresponders the increase was significant. In nonresponders, sputum eosinophils also significantly increased after ozone. There was a positive correlation between ozone-induced FEV1 fall and increase in sputum neutrophils. No difference in functional or inflammatory response to ozone was observed between subjects with or without the combination of NQO1wt- GSTM1null genotypes.

Conclusions

Markers of neutrophilic inflammation and oxidative stress increase also in asthmatic subjects not responding to ozone. A greater functional response to ozone is associated with greater neutrophil airway recruitment in asthmatic subjects.     

Haemophilus influenzae infection drives IL-17-mediated neutrophilic allergic airways disease

A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12-15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses.     

Inhibition of neutrophil elastase attenuates airway hyperresponsiveness and inflammation in a mouse model of secondary allergen challenge: neutrophil elastase inhibition attenuates allergic airway responses

Background

Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice.

Methods

BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge.

Results

Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-β1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice.

Conclusion

These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.     

Sputum IgE and Cytokines in Asthma: Relationship with Sputum Cellular Profile

Background

Local IgE production may play a role in asthma pathogenesis. The aim of the study was to assess sputum total IgE and cytokines in asthmatics according to sputum cellular phenotype.

Methods

We studied 122 subjects including 22 non atopic healthy subjects, 41 eosinophilic (sputum eosinophils ≥3%), 16 neutrophilic (sputum neutrophils >76%) and 43 pauci-granulocytic asthmatics (sputum eosinophils <3% and sputum neutrophils ≤76%) recruited from the asthma clinic at CHU Liege.Sputum supernatant total IgE (tIgE) was measured by ImmunoCAP and sputum supernatant cytokines (IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ and TNF-α) were measured with the Luminex xMAP Technology by using commercially available Fluorokine MAP kits.

Results

After concentrating sputum samples, total IgE was detectable in the majority of subjects. Sputum IgE was raised in asthmatics when compared to healthy subjects. Overall, asthmatics did not significantly differ from healthy subjects with respect to cytokine levels. The eosinophilic asthma phenotype, however, was characterised by raised sputum tIgE, IL-5 and IL-13 compared to healthy subjects (p<0.001, p<0.001 and p<0.05 respectively) and pauci-granulocytic asthma (p<0.01, p<0.001 and p<0.05 respectively) and raised IL-5 compared to neutrophilic asthma (p<0.01). When patients were classified according to sputum IgE levels, it appeared that IL-5, IL-6, IL-17 and TNF-α sputum supernatant levels were raised in the “IgE high” asthmatics (IgE ≥0.1 kU/l) when compared to “IgE low” asthmatics (IgE<0.1 kU/l).

Conclusion

The eosinophilic asthma phenotype was associated with raised sputum IgE and a Th2 cytokine profile. Raised sputum IgE was associated with a heterogeneous cytokine overproduction.     

Analysis of a Panel of 48 Cytokines in BAL Fluids Specifically Identifies IL-8 Levels as the Only Cytokine that Distinguishes Controlled Asthma from Uncontrolled Asthma,and Correlates Inversely with FEV1

We sought to identify cells and cytokines in bronchoalveolar lavage (BAL) fluids that distinguish asthma from healthy control subjects and those that distinguish controlled asthma from uncontrolled asthma. Following informed consent, 36 human subjects were recruited for this study. These included 11 healthy control subjects, 15 subjects with controlled asthma with FEV₁≥80% predicted and 10 subjects with uncontrolled asthma with FEV₁ <80% predicted. BAL fluid was obtained from all subjects. The numbers of different cell types and the levels of 48 cytokines were measured in these fluids. Compared to healthy control subjects, patients with asthma had significantly more percentages of eosinophils and neutrophils, IL-1RA, IL-1α, IL-1β, IL-2Rα, IL-5, IL-6, IL-7, IL-8, G-CSF, GROα (CXCL1), MIP-1β (CCL4), MIG (CXCL9), RANTES (CCL5) and TRAIL in their BAL fluids. The only inflammatory markers that distinguished controlled asthma from uncontrolled asthma were neutrophil percentage and IL-8 levels, and both were inversely correlated with FEV₁. We examined whether grouping asthma subjects on the basis of BAL eosinophil % or neutrophil % could identify specific cytokine profiles. The only differences between neutrophil-normal asthma (neutrophil≤2.4%) and neutrophil-high asthma (neutrophils%>2.4%) were a higher BAL fluid IL-8 levels, and a lower FEV₁ in the latter group. By contrast, compared to eosinophil-normal asthma (eosinophils≤0.3%), eosinophil-high asthma (eosinophils>0.3%) had higher levels of IL-5, IL-13, IL-16, and PDGF-bb, but same neutrophil percentage, IL-8, and FEV₁. Our results identify neutrophils and IL-8 are the only inflammatory components in BAL fluids that distinguish controlled asthma from uncontrolled asthma, and both correlate inversely with FEV₁.     

Non-Eosinophilic Nasal Polyps Shows Increased Epithelial Proliferation and Localized Disease Pattern in the Early Stage

BackgroundNon-eosinophilic nasal polyps (NPs) show less inflammatory changes and are less commonly associated with lower airway inflammatory disorders such as asthma, compared with eosinophilic NPs. However, the development of non-eosinophilic NPs which is a predominant subtype in Asian population still remains unclear.MethodsA total of 81 patients (45 with non-eosinophilic NPs and 36 with eosinophilic NPs) were enrolled. Clinical information and computed tomography (CT), endoscopic, and histological findings were investigated. Tissue samples were analyzed for total IgE levels and for mRNA expression levels of interleukin (IL)-4, IL–5, IL–13, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-17A, IL–22, IL-23p19, transforming growth factor (TGF)-β1, TGF-β2, TGF-β3, and periostin. Immunostaining assessment of Ki–67 as a proliferation marker was performed.ResultsWe found that epithelial in-growing patterns such as pseudocysts were more frequently observed in histological and endoscopic evaluations of non-eosinophilic NPs, which was linked to increase epithelial staining of Ki–67, a proliferating marker. Eosinophilic NPs were characterized by high infiltration of inflammatory cells, compared with non-eosinophilic NPs. To investigate the developmental course of each subtype, CT was analyzed according to CT scores and subtypes. Non-eosinophilic NPs showed more localized pattern and maxillary sinus involvement, but lesser olfactory involvement in early stage whereas eosinophilic NPs were characterized by diffuse ethmoidal and olfactory involvement. In addition, high ethmoidal/maxillary (E/M) CT scores, indicating ethmoidal dominant involvement, were one of surrogate markers for eosinophilic NP. E/M CT scores was positively correlated with levels of T_H2 inflammatory markers, including IL–4, IL–5, periostin mRNA expression and total IgE levels in NPs, whereas levels of the T_H1 cytokine, IFN- γ were inversely correlated. Moreover, if the combinatorial algorithm meet the three of the four markers, including IL–5 (<2.379), periostin (<3.889), IFN-γ (>0.316), and E/M ratio (<2.167), non-eosinophilic CRSwNP are diagnosed with a sensitivity of 84.4% and a specificity of 84.8%.ConclusionHistologic, immunologic and clinical data suggest that non-eosinophilic NPs showed enhanced epithelial alteration and more localized maxillary involvement. Combination of cutoff value on IL–5, periostin, IFN-γ, and E/M scores may be one of surrogate markers for non-eosinophil NP subtype.     

Inhaled Lead Exposure Affects Tracheal Responsiveness and Lung Inflammation in Guinea Pigs During Sensitization

Total and differential white blood cells (WBC), and cytokines, levels in serum were examined in guinea pigs exposed to inhaled lead acetate. Different groups of guinea pigs including: control (group C), sensitized group (group S), and exposed animals to aerosol of three lead concentrations during sensitization (n?=?6 for each group) were studied. Total and differential WBC counts of lung lavage, serum cytokine (IFNγ and IL-4), levels and tracheal responsiveness to methacholine and ovalbumin were measured. All measured values were significantly increased except for IFNγ/IL-4 ratio which was significantly decreased in nonexposed sensitized and those exposed to all lead concentrations compared to control group (p?<?0.05 to p?<?0.001). Most measured values in animals exposed to higher lead concentration were also significantly higher than group S except for tracheal responsiveness to methacholine and lymphocyte count. Lead concentration significantly increased in lung tissues of animals exposed to all three lead concentrations (p?<?0.001 for all cases). These results showed that lead exposure during sensitization can induce greater increase in tracheal responsiveness, total WBC, eosinophil, neutrophil, and basophil counts as well as serum level of IL-4. It can also cause a decrease in lymphocyte count, IFNγ level, and IFNγ/IL-4 ratio especially in its high concentration. Therefore inhaled lead exposure may cause increased severity of asthma during development of the disease.     

百令胶囊联合罗氟司特治疗老年支气管哮喘的效果及对免疫功能的影响

目的:研究百令胶囊联合罗氟司特治疗老年支气管哮喘的效果及对免疫功能的影响。方法:选择2018年3月~2019年3月我院第六派驻门诊部收治的110例老年支气管哮喘患者,随机分为两组,每组各55例。对照组患者口服罗氟司特片,每次500 mg,每天1次。观察组患者联合服用百令胶囊,每次5粒,每天3次。比较两组的临床控制率,治疗前后两组诱导痰中炎症因子、中性粒细胞、呼出气一氧化氮(fractional exhaled nitric oxide,FeNO)水平和免疫功能的变化。结果:治疗后,观察组的临床控制率为70.91%(39/55),明显高于对照组[40.00%(22/55)](P<0.05);两组的CD4+、CD4+/CD8+和CD3+均较治疗前明显升高,且观察组的CD4+、CD4+/CD8+和CD3+明显高于对照组(P<0.05);两组患者诱导痰中中性粒细胞绝对值、中性粒细胞百分比、白细胞介素-4(interleukin-4,IL-4)、FeNO、白细胞介素-17(interleukin-17,IL-17)均较治疗前明显降低(P<0.05),诱导痰中的干扰素-γ(Interferon gamma,IFN-γ)均较治疗前明显升高(P<0.05),且观察组诱导痰中的组中性粒细胞绝对值、中性粒细胞百分比、IL-4、FeNO、IL-17明显低于对照组(P<0.05),诱导痰中的IFN-γ均明显高于对照组(P<0.05)。结论:百令胶囊联合罗氟司特对老年支气管哮喘患者具有显著的疗效,其机制可能与抑制中性粒细胞的激活、调节T淋巴细胞亚群的平衡和抑制相关炎症因子的释放相关。     

Potentially pathogenic bacteria cultured from the sputum of stable asthmatics are associated with increased 8-isoprostane and airway neutrophilia

Abstract

Potential bacterial pathogens are found in the airways in several diseases that are associated with neutrophilic inflammation. The aim of this study was to characterize subjects with stable asthma, with no symptoms of respiratory infection, to assess whether key potentially pathogenic bacteria were present in significant quantities in the airways and to correlate this with the pattern of airway inflammation and oxidative stress. Subjects with stable asthma (n = 115) and healthy controls (n = 8) underwent clinical assessment, including hypertonic saline challenge combined with sputum induction. A significant load of potentially pathogenic bacteria (> 10⁶ cfu/mL) was cultured from the sputum of 17 (15%) subjects with stable asthma and was associated with higher total cell counts, proportion and number of neutrophils, sputum IL-8 and 8-isoprostane concentrations. The role of bacteria in potentiating neutrophilic asthma warrants further investigation. Therapies such as antibiotic and antioxidant treatment may be most effective in this sub-group of patients.     

Leukotrienes cause eosinophil emigration into conjunctival tissue

The ability of LTB4, LTC4, the 5S,6R and 5R,6S LTD4 stereoisomers, and LTE4 to evoke leukocyte infiltration into the conjunctiva was demonstrated in the guinea pig by histological and light microscopy techniques. LTD4 and LTE4 demonstrated a dose-dependent and predominantly eosinophilic infiltrate over the selected dose range (10 ng to 1000 ng), while there was only a minimal response to LTC4. LTB4 produced marked eosinophil infiltrates only at the highest dose; scattered neutrophil infiltrates were also noted at the high dose of LTB4. The 5R,6S LTD4 stereoisomer did not evoke any leukocyte infiltration. The SRS-A antagonist, FPL 55712, abolished peptidoleukotriene-induced eosinophil emigration, and indomethacin pre-treatment had no inhibitory effect, indicating direct mediation of this response by LTs. Histamine caused a comparable eosinophilia over a dose range of 10 micrograms to 1000 micrograms. LT-induced eosinophil emigration was directed to the conjunctival epithelium; the cells appeared intact and no tissue damage was observed. These results may have relevance in the areas of allergic conjunctivitis and asthma research.     

Cytokine-directed therapies for the treatment of chronic airway diseases

Multiple cytokines play a critical role in orchestrating and perpetuating inflammation in asthma and chronic obstructive pulmonary disease (COPD) and several specific cytokine and chemokine inhibitors now in development as future therapy for these diseases. Anti-IL-5 antibody markedly reduces peripheral blood and airway eosinophils, but does not appear to be effective in symptomatic asthma. Inhibition of IL-4 despite promising early results in asthma has been discontinued and blocking IL-13 might be more effective. Inhibitory cytokines, such as IL-10, interferons and IL-12 are less promising, as systemic delivery produces side effects. Inhibition of TNF-alpha may be useful in severe asthma and for treating severe COPD with systemic features. Many chemokines are involved in the inflammatory response of asthma and COPD and several small molecule inhibitors of chemokine receptors (CCR) are in development. CCR3 antagonists (which block eosinophil chemotaxis) and CXCR2 antagonists (which block neutrophil and monocyte chemotaxis) are in clinical development for asthma and COPD, respectively. Because so many cytokines are involved in asthma, drugs that inhibit the synthesis of multiple cytokines may prove to be more useful; several such classes of drug are now in clinical development and any risk of side effects with these non-specific inhibitors may be reduced by the inhaled route.     

Airway exposure levels of lipopolysaccharide determine type 1 versus type 2 experimental asthma

Allergic asthma is characterized by airway inflammation initiated by adaptive immune responses to aeroallergens. Recent data suggest that severe asthma may be a different form of asthma rather than an increase in asthma symptoms and that innate immune responses to LPS can modulate adaptive immune responses to allergens. In this study, we evaluated the hypothesis that airway exposure to different doses of LPS induces different form of asthma. Our study showed that neutrophilic inflammation and IFN-gamma expression were higher in induced sputum from severe asthma patients than from mild to moderate asthmatics. Animal experiments indicated that allergen sensitization with low-dose LPS (0.1 microg) induced type 2 asthma phenotypes, i.e., airway hyperresponsiveness, eosinophilic inflammation, and allergen-specific IgE up-regulation. In contrast, allergen sensitization with high-dose LPS (10 microg) induced asthma phenotypes, i.e., airway hyperresponsiveness and noneosinophilic inflammation that were not developed in IFN-gamma-deficient mice, but unaffected in the absence of IL-4. During the allergen sensitization period, TNF-alpha expression was found to be enhanced by both low- and high-dose LPS, whereas IL-12 expression was only enhanced by high-dose LPS. Interestingly, the asthma phenotypes induced by low-dose LPS, but not by high-dose LPS, were completely inhibited in TNF-alpha receptor-deficient mice, whereas the asthma phenotypes induced by high-dose LPS were abolished in the homozygous null mutation of the STAT4 gene. These findings suggest that airway exposure levels of LPS induces different forms of asthma that are type 1 and type 2 asthma phenotypes by high and low LPS levels, respectively.     

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-1β 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.