Number Of Neutrophils In Airways Research Articles

G-CSFR antagonism reduces neutrophilic inflammation during pneumococcal and influenza respiratory infections without compromising clearance

Excessive neutrophilic inflammation can contribute to the pathogenesis of pneumonia. Whilst anti-inflammatory therapies such as corticosteroids are used to treat excessive inflammation, they do not selectively target neutrophils and may compromise antimicrobial or antiviral defences. In this study, neutrophil trafficking was targeted with a granulocyte-colony stimulating factor receptor monoclonal antibody (G-CSFR mAb) during Streptococcus pneumoniae (serotype 19F) or influenza A virus (IAV, strain HKx31) lung infection in mice. Firstly, we demonstrated that neutrophils are indispensable for the clearance of S. pneumoniae from the airways using an anti-Ly6G monoclonal antibody (1A8 mAb), as the complete inhibition of neutrophil recruitment markedly compromised bacterial clearance. Secondly, we demonstrated that G-CSF transcript lung levels were significantly increased during pneumococcal infection. Prophylactic or therapeutic administration of G-CSFR mAb significantly reduced blood and airway neutrophil numbers by 30–60% without affecting bacterial clearance. Total protein levels in the bronchoalveolar lavage (BAL) fluid (marker for oedema) was also significantly reduced. G-CSF transcript levels were also increased during IAV lung infection. G-CSFR mAb treatment significantly reduced neutrophil trafficking into BAL compartment by 60% and reduced blood neutrophil numbers to control levels in IAV-infected mice. Peak lung viral levels at day 3 were not altered by G-CSFR therapy, however there was a significant reduction in the detection of IAV in the lungs at the day 7 post-infection phase. In summary, G-CSFR signalling contributes to neutrophil trafficking in response to two common respiratory pathogens. Blocking G-CSFR reduced neutrophil trafficking and oedema without compromising clearance of two pathogens that can cause pneumonia.

Granulocyte colony-stimulating factor blockade enables dexamethasone to inhibit lipopolysaccharide-induced murine lung neutrophils.

Glucocorticoids promote neutrophilic inflammation, the mechanisms of which are poorly characterized. Using a lipopolysaccharide (LPS)-induced acute murine lung injury model, we determined the role of granulocyte colony-stimulating factor (G-CSF) in mouse lung neutrophil numbers in the absence and presence of dexamethasone, a potent glucocorticoid. G-CSF was blocked using a neutralizing antibody. Airway neutrophil numbers, cytokine levels, and lung injury parameters were measured. Glucocorticoid treatment maintained LPS-induced airway G-CSF while suppressing TNF and IL-6. The addition of anti-G-CSF antibodies enabled dexamethasone to decrease airway G-CSF, neutrophils, and lung injury scores. In LPS-challenged murine lungs, structural cells and infiltrating leukocytes produced G-CSF. In vitro using BEAS 2B bronchial epithelial cells, A549 lung epithelial cells, human monocyte-derived macrophages, and human neutrophils, we found that dexamethasone and proinflammatory cytokines synergistically induced G-CSF. Blocking G-CSF production in BEAS 2B cells using shRNAs diminished the ability of BEAS 2B cells to protect neutrophils from undergoing spontaneous apoptosis. These data support that G-CSF plays a role in upregulation of airway neutrophil numbers by dexamethasone in the LPS-induced acute lung injury model.

Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis.

Nitric oxide (NO) contributes to protection from tuberculosis (TB). It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis (Mtb) growth, which prevents subsequent pathological inflammation. In contrast, we report NO primarily protects mice by repressing an interleukin-1 and 12/15-lipoxygenase dependent neutrophil recruitment cascade that promotes bacterial replication. Using Mtb mutants as indicators of the pathogen's environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports Mtb growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes TB in patients. The human 12/15 lipoxygenase ortholog, ALOX12, is expressed in cavitary TB lesions, the abundance of its products correlate with the number of airway neutrophils and bacterial burden, and a genetic polymorphism that increases ALOX12 expression is associated with TB risk. These data suggest that Mtb exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion.

Therapeutic implications of 'neutrophilic asthma'.

This review examines the association between airway neutrophilia and severe asthma, potential mechanisms, and the effect on asthma control of therapies directed at reducing airway neutrophil numbers or activity. The majority of studies that observe an association between airway neutrophilia and severe asthma are cross-sectional in nature, and the intensity of neutrophilia is low and may be a reflection of the age of the patients, effect of tobacco smoke exposure, or the high doses of corticosteroids used to treat their asthma. There may be a small proportion of patients who may have abnormal innate immune responses that may lead to airway neutrophilia. However, these neutrophils may not be any more activated than in patients with milder asthma. Novel strategies using small molecule antagonists against the interleukin-8 receptor, CXCR2, are able to reduce airway neutrophilia, and their clinical efficacies are being investigated. Although cross-sectional studies suggest that airway neutrophilia may be observed in some patients with severe asthma, it is not clearly established if this is a consequence of treatment with corticosteroids or if it contributes directly to asthma pathobiology and severity. New therapies such as anti-CXCR2 provide an opportunity to investigate the contribution of neutrophils to asthma severity.

Increased neutrophil migration in smokers with or without chronic obstructive pulmonary disease

The number of airway neutrophils is increased in chronic obstructive pulmonary disease (COPD), and this may have a central pathophysiological role in the disease. In addition, activation of neutrophils increases their migration into sites of injury. We hypothesize that circulating neutrophils are activated in smokers. Peripheral blood neutrophils were isolated from healthy non-smokers (n = 15), and smokers with (n = 15) or without COPD (n = 15), who were matched with regard to cumulative tobacco exposure, and chemotactic responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL-8, CXCL8) and leukotriene B(4) (LTB(4)) were assessed using the ChemoTx System (Neuro Probe Inc., Gaithersburg, MD, USA). Serum tumour necrosis factor-α (TNF-α) concentrations were measured by ELISA. Surface expression of the neutrophil activation marker, CD11b, was measured by flow cytometry. The chemotactic response to CXCL8 was increased in smokers with or without COPD (P < 0.05). Migration towards LTB(4) was increased in smokers without COPD compared with non-smokers (P < 0.05), whereas there was no difference in fMLP-induced chemotaxis between the groups. There was a correlation between serum TNF-α levels and migration induced by IL-8 (Rho = 0.442; P = 0.038) and LTB(4) (Rho = 0.428; P = 0.044) in the smokers. Furthermore, there was a tendency towards higher CD11b expression in the COPD group (P = 0.057). Chemotaxis of circulating neutrophils towards CXCL8, and partly towards LTB(4), is increased in smokers, indicating a systemic influence of smoking on cell activation, irrespective of the presence of airflow limitation. The relationship between TNF-α and chemotactic response suggests that TNF-α is involved in neutrophil activation, resulting in enhanced migration.

Persistence of systemic inflammation in COPD in spite of smoking cessation

Cigarette smoking, which exposes the lung to high concentrations of reactive oxygen species (ROS), is the major risk factor for chronic obstructive pulmonary disease (COPD) [1]. ROS are responsible for lipid peroxidation, protein oxidation, DNA alterations, and cell damage. Coupled with oxidant/antioxidant imbalance, these changes lead to lung inflammatory responses through multiple pathways, among which the activation of stress kinases (c-Jun activated kinase, extracellular signal-regulated kinase, p38 kinase) and redox-sensitive transcription factors, such as nuclear factor (NF)-kappaB and activator protein-1, resulting in the expression of kinase- and NF-κB-dependent proinflammatory cytokines and chemokines [2]. COPD is related to smoking habit in the majority of cases, and smoking cessation is the single most beneficial and effective way to reduce COPD morbidity, hospital admissions and disease progression [1]. However, persistent inflammation in the airways of COPD subjects may continue after smoking cessation, as shown by studies showing a similar number of airway inflammatory cells, including CD4+ and CD8+ lymphocytes, in the bronchial biopsies of both current smokers and ex-smokers with COPD [3], and by other studies analyzing the composition of induced sputum, which showed a persistent increase in the number of airway neutrophils and lymphocytes one year after smoking cessation [4]. A persistent overall burden of non invasive markers of oxidative stress has also been found in the airways after smoking cessation [5]. Also systemic markers of inflammation were found elevated in the serum of COPD patients [6,7]. Taken together, these studies indicate that, in COPD patients, ongoing inflammation is persistent in spite of smoking cessation: this phenomenon is considered one of the determinants of the progressive decline in lung function. This issue of Multidisciplinary Respiratory Medicine features an article by Serapinas et al. [8] on the presence of markers of systemic inflammation in a sample of 320 COPD patients according to their smoking status. The authors devoted their attention to the serum concentrations of the following set of oxidant markers: C-reactive protein (CRP), alpha-1 antitrypsin (AAT), tumor necrosis factor-α (TNF-α), and soluble tumor necrosis factor receptor (sTNFR)-1 and sTNFR-2. The markers were analyzed in relation to current smoking (n = 202) and ex-smoking (n = 61), as well never-smoker (n = 57) status. Mean (SD) forced expiratory volume in one second/forced vital capacity (FEV1/FVC) (%) was 54.8 (10.7), while mean (SD) pack/years in smokers and ex-smokers was 22.7 ± 12.9. The main result of the study was that AAT, CRP and sTNFR-1 concentrations were significantly increased in both smokers and ex-smokers as compared to never-smokers. This study adds to the number of reports showing the presence of systemic and airway inflammation in COPD patients, irrespective of whether they were current or ex-smokers. Although inflammation is also present as a consequence of smoking in the airways of asymptomatic smokers, a significant decrease of oxidant markers occurs after smoking cessation in these subjects [4]. It is not known why - at variance with what occurs in asymptomatic smokers - inflammation persists after quitting smoking in COPD patients [1]. One of the leading explanations is the altered regulation of inflammatory gene expression, even though epigenetic mechanisms are implicated too. In a recent study it has been shown that smoking per se modulates the expression of genes from all the inflammatory functional groups except for the matrix metalloproteinases group [10]. The inflammation related genes (except TNF-a) were found to be up-regulated, while genes from the growth factor/receptor group, adhesion molecules and vessel tone/maintenance factors were down-regulated in smokers. It is notable that all of these genes exhibited a similar profile in patients with moderate COPD, suggesting a crucial role of cigarette smoking in the genesis of these changes. However, one of the most significant changes associated with COPD was the increased expression of matrix metalloproteinases [10]. Although the COPD puzzle is still far from being solved, multidisciplinary research linking the different expertise of epidemiological, biological, translational and clinical nature is believed to be the best response to such a difficult task.

A Critical Role for C5L2 in the Pathogenesis of Experimental Allergic Asthma

The complement fragment C5a plays dual roles in the development of experimental allergic asthma. It protects from pulmonary allergy by a regulatory effect on dendritic cells during allergen sensitization, but is proallergic during the effector phase. C5a can bind to two distinct receptors (i.e., C5a receptor and C5a receptor-like 2 [C5L2]). The functional role of C5L2 in vivo remains enigmatic. In this study, we show in two models of OVA- and house dust mite (HDM)-induced experimental allergic asthma that C5L2-deficient mice are protected from the development of airway hyperresponsiveness, Th2 cytokine production, eosinophilic airway inflammation, serum IgE, or mucus production. Surprisingly, HDM-induced experimental asthma in C5L2-deficient mice was associated with increased pulmonary IL-17A production and increased airway neutrophil numbers. To directly assess the role of C5L2 on myeloid dendritic cells (mDCs) during allergen sensitization, we performed single or repeated adoptive transfers of C5L2-deficient mDCs into wild-type mice. HDM-pulsed C5L2-deficient mDCs induced strong Th2 cytokine production, which was associated with marked IFN-γ and IL-17A production, decreased eosinophil numbers, and reduced IgE production as compared with HDM-pulsed mDCs from wild-type mice. HDM stimulation of C5L2(-/-) mDCs in vitro resulted in production of Th17-promoting cytokine IL-23, which was absent in wild-type mDCs. Our findings suggest that C5L2 acts at the mDC/T cell interface to control the development of Th1 and Th17 cells in response to airway HDM exposure. Furthermore, it drives Th2 immune responses independent of mDCs, suggesting a complex role for C5L2 in the development of experimental allergic asthma.

Reactivity of Airway Phagocytes during the Development of Acute Pneumonia under Conditions of Stimulation of Mononuclear Phagocyte System with Zymosan

Pneumonia was induced in (CBA x C57Bl)F1 mice under conditions of stimulation of the mononuclear phagocyte system with zymosan. The number of neutrophils in airways increased after 3 days; by day 14, the number of cells in the bronchoalveolar lavage fluid further increased due to migration of macrophages. After zymosan prestimulation, the number and functional activity of neutrophils during the early period of inflammation (3 days) did not change, but the increase in phagocytic activity of macrophages was inhibited by 20%. By day 14, the effect of prestimulation manifested in 4.5-fold decreased capacity of neutrophils and macrophages to reduce NBT.

Activation of Neurokinin-1 Receptors during Ozone Inhalation Contributes to Epithelial Injury and Repair

We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2'-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress-positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

High-dose oral N -acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis

Neutrophilic airway inflammation is a hallmark of cystic fibrosis (CF). As high oxidant producers, airway neutrophils contribute largely to the systemic redox imbalance seen in CF. In turn, this chronic and profound imbalance can impact circulating neutrophils before their migration into airways. Indeed, in 18 CF patients with stable disease, blood neutrophils were readily deficient in the pivotal antioxidant glutathione (P = 0.003, compared with 9 healthy controls). In a phase 1 study, this deficiency was improved (P = 0.025) by the glutathione prodrug N-acetylcysteine, given orally in high doses (0.6 to 1.0 g three times daily, for 4 weeks). This treatment was safe and markedly decreased sputum elastase activity (P = 0.006), the strongest predictor of CF pulmonary function. Consistently, neutrophil burden in CF airways was decreased upon treatment (P = 0.003), as was the number of airway neutrophils actively releasing elastase-rich granules (P = 0.005), as measured by flow cytometry. Pulmonary function measures were not improved, as expected with short-term treatment. After excluding data from subjects without baseline airway inflammation, positive treatment effects were more pronounced and included decreased sputum IL-8 levels (P = 0.032). Thus, high-dose oral N-acetylcysteine has the potential to counter the intertwined redox and inflammatory imbalances in CF.

Neutrophils Regulate Airway Responses in a Model of Fungal Allergic Airways Disease

Neutrophils infiltrate airway walls in patients with allergic airway diseases and in animal models of these illnesses, but their contribution to the pathogenesis of airway allergy is not established. We hypothesized that, in a mouse model of airway allergy to the ubiquitous environmental mold, Aspergillus fumigatus, airway neutrophils contribute to disease severity. Ab-mediated neutrophil depletion resulted in reduced airway hyperresponsiveness and remodeling, whereas conditional transgenic overexpression of the neutrophil chemotactic molecule, CXCL1, in airway walls resulted in worsened allergic responses. This worsened phenotype was associated with a marked increase in the number of airway neutrophils but not other lung leukocytes, including eosinophils and lymphocyte subsets, and depletion of neutrophils in sensitized mice with transgenic overexpression of CXCL1 resulted in attenuated airway responses. The number of lung neutrophils correlated with lung matrix metalloproteinase 9 (MMP-9) activity both in the context of neutrophil depletion and with augmented neutrophil recruitment to the airways. Although wild-type and MMP-9-deficient neutrophils homed to the inflamed airways to a similar extent, transfer of wild-type, but not MMP-9-deficient, neutrophils to MMP-9-deficient animals resulted in augmented allergic airway responses. Taken together, these data implicate neutrophils in the pathogenesis of fungal allergic airway disease.

Interleukin-17 as a Recruitment and Survival Factor for Airway Macrophages in Allergic Airway Inflammation

Recent data indicate that the proinflammatory cytokine, interleukin (IL)-17, stimulates certain effector functions of human macrophages. We evaluated whether IL-17 mediates allergen-induced accumulation of airway macrophages and, if so, whether such an effect relates to the control of macrophage recruitment and survival. BALB/c mice were sensitized and challenged with ovalbumin. Three hours before challenge an anti-mouse IL-17 mAb (a-IL-17) was administered. Sampling was conducted 24 h after the allergen challenge. In vitro chemotaxis assay for blood monocytes and culture of airway macrophages, immunocytochemistry for Fas-antigen, and matrix metalloproteinase-9 (MMP-9) were used to determine the effect of IL-17 on the recruitment, survival, and activity of airway macrophages. A-IL-17 reduced the number of airway neutrophils and macrophages after allergen challenge. In vitro, recombinant IL-17 induced migration of blood monocytes and prolonged survival of airway macrophages. A-IL-17 also increased the expression of Fas-antigen in airway macrophages in vivo. Finally, the expression of MMP-9 by airway neutrophils and macrophages in vivo was downregulated by a-IL-17. This study indicates that endogenous IL-17 mediates the accumulation of macrophages during allergen-induced airway inflammation. IL-17 exerts its effects by acting directly on airway macrophages by promoting their recruitment and survival. Furthermore, IL-17 is involved in controlling the proteolytic activity of macrophages and neutrophils in allergen-induced airway inflammation.

Endotoxin contamination contributes to the pulmonary inflammatory and functional response to Aspergillus fumigatus extract inhalation in heaves horses.

Mould extract inhalation challenges have been used extensively in the investigation of heaves. Such challenges have induced pulmonary neutrophilic inflammation and dysfunction, consistent with, but less severe than the natural disease. However, the method of mould extract production is likely to result in endotoxin contamination. To investigate whether insufficient dose delivery was responsible for the shortfall in response to inhaled extract compared with natural disease, and whether endotoxin contamination of mould extract contributed to the pulmonary inflammation and dysfunction. We measured the response of six heaves horses following inhalation of saline (placebo) and three doses of Aspergillus fumigatus extract. We then compared the response of six heaves horses to A. fumigatus extract inhalation before and after lipopolysaccharide (LPS) depletion. Inhalation challenge with 0.5, 1.6 and 5 mg of A. fumigatus extract resulted in a significant increase in bronchoalveolar lavage fluid (BALF) neutrophil ratio when compared with saline inhalation. Only 1.6 and 5 mg extract inhalation resulted in significant lung dysfunction compared with saline. There was no significant difference between 1.6 and 5 mg extract inhalation with respect to airway neutrophil numbers or lung function, suggesting a plateau in both measured responses. LPS depletion of 1.6 mg A. fumigatus extract resulted in a significant reduction in airway neutrophil numbers and increase in arterial oxygen tension. There was no significant difference between saline and the LPS-depleted A. fumigatus extract challenges with respect to neutrophil count and lung function. The reduction in airway neutrophil numbers was greater than would be predicted by extrapolation from previously reported soluble LPS dose-response inhalation experiments. This study supports a role for other inhalants, in addition to soluble components of A. fumigatus, in the aetiopathogenesis of heaves. Also the amplification in response to LPS when inhaled with A. fumigatus extract, suggests that the role of inhaled endotoxin in the pulmonary inflammation and dysfunction in naturally occurring heaves may currently be underestimated.

Airway neutrophils and interleukin-17.

It is well known that exacerbations of obstructive airways disease such as asthma and chronic obstructive pulmonary disease are associated with an increased number of neutrophils in the airways. However, the mechanisms behind this phenomenon are poorly understood. There is in vivo experimental evidence that the number of airway neutrophils is controlled by certain T-lymphocytes, but the mediators responsible for this lymphocyte-related neutrophilia have not yet been identified. In this review, novel evidence that the T-lymphocyte-related cytokine interleukin (IL)-17 can link the activation of certain T-lymphocytes to the recruitment and activation of airway neutrophils is described. The IL-17-induced neutrophil recruitment is mediated via induced CXC chemokine release through steroid-sensitive mechanisms and is modulated by release of endogenous tachykinins. These effects of IL-17 are potentiated by other pro-inflammatory cytokines such as (IL-1beta) and tumour necrosis factor-alpha. Clinical studies are needed to evaluate whether or not targeting these mechanisms can provide a useful pharmacotherapeutical approach against exaggerated mobilization of neutrophils in obstructive airways disease.

Tracheal Aspirates in Long-term Mechanically Ventilated Patients: A Human Model of Gram-Negative Infection and Airway Inflammation

It is well known that patients requiring long-term mechanical ventilation and tracheostomy have nearly universal airway colonization with Gram-negative organisms. However, useful parameters to objectively describe the airway inflammation associated with airway instrumentation and colonization have not been well define. In our respiratory care unit, patients who are medically stable except for ventilator dependence are readily available for longitudinal assessment of airway secretions and therefore provide a unique population for studying airway inflammation and infection. To quantitate production of respiratory secretions, we instituted a uniform protocol of suctioning over a 6-h period. Further, we devised a method of dilution and homogenization of tracheal aspirates that permits reproducible intrasample total cell counts (coefficient of variation, 4.6%). With these techniques, patients were then studied serially over a 4- to 7-week period. Total cell count, inflammatory cell differential, and two indices of airway inflammation, human neutrophil elastase (HLE) and soluble-intercellular adhesion molecule-1 (sICAM-1) studied in the sol phase of secretions were monitored. The mean total cell count was 42.2 x 10(6) cells per gram of secretions when patients were clinically stable and not receiving antibiotics. The average differential was neutrophils 69.9%, macrophages 26.9%, and lymphocytes 2.8%. Mean active HLE was 35.6 micrograms/mL and mean sICAM-1 was 83 ng/mL. Six patients during the period of observation received intravenous oral or aerosolized antibiotics for tracheobronchitis. A threefold drop in volume of secretions was measured (p < 0.018). The total cell count and percent neutrophils decreased from 76.4 x 10(6)/g of sputum to 54.9 x 10(6) and 72.2 to 54.9%, respectively. While these changes were not statistically significant, the absolute number of airway neutrophils over the 6 h decreased sevenfold (p < 0.014). Similarly sICAM-1 burden (micrograms per 6-h period) also decreased significantly (p < 0.034). These patients provide a unique human model for future studies specifically designed to assess the effect of novel modalities of anti-inflammatory and antimicrobial agents on respiratory secretions.