Effect of cystic fibrosis exacerbations on neutrophil function

doi:10.1016/j.intimp.2004.11.007

International Immunopharmacology

Volume 5, Issue 3, March 2005, Pages 601-608

https://doi.org/10.1016/j.intimp.2004.11.007 Get rights and content

Abstract

In cystic fibrosis (CF), inflammation is caused by persistent bacterial infection from Pseudomonas aeruginosa and Burkholderia cenocepacia in the lung and is characterised by the persistent infiltration of massive numbers of neutrophils which leads to lung injury. The aim of this present study was to investigate the effects of CF exacerbations on the reactivity of peripheral blood neutrophils compared to data from a normal healthy control population. Peripheral blood neutrophils were isolated from control subjects and CF patients before and after an exacerbation of their lung disease. Isolated neutrophils were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA) and the rate of superoxide generation and elastase activity measured and compared with neutrophils from healthy age-matched controls. Neutrophils from CF patients spontaneously generated higher levels of superoxide after resolution of the exacerbation compared to control neutrophils. The stimulated generation of superoxide from control neutrophils was not significantly different from neutrophils isolated from CF patients either before or after resolution of the CF exacerbation. Neutrophils from CF patients spontaneously released more elastase than control neutrophils but released less elastase than control neutrophils in response to fMLP. The stimulated release of elastase from neutrophils was not significantly different before compared to after resolution of the exacerbation. Neutrophils from CF patients displayed a different pattern of response than those from control subjects; however, CF exacerbations did not appear to modulate neutrophil function.

Introduction

Cystic fibrosis (CF) is characterised by chronic progressive bronchiectasis, exocrine pancreatic insufficiency, sterility in males, and abnormally high concentrations of sodium chloride in the sweat. Water reabsorption from the CF airway lumen outweighs limited Cl⁻ secretion resulting in the airway secretions becoming viscid and clearance of secretions is impaired causing airway obstruction [1]. This is further compounded by colonisation of the airway with organisms such as Pseudomonas aeruginosa (P. aeruginosa) and Burkholderia cenocepacia (B. cenocepacia) which are associated with a poor prognosis [2], [3].

Inflammation caused by infection in the lungs plays a central role in the vicious cycle that leads to lung destruction. The most characteristic feature of inflammation in the CF lung is the persistent infiltration of massive numbers of neutrophils into the airways [4], [5]. Neutrophils in the airways undergoing necrosis in situ are a major source of DNA, which makes CF sputum so tenacious [5]. The excessive accumulation of activated neutrophils in the lungs can lead to lung damage [6]. Proinflammatory chemoattractants have been found to be elevated in bronchoalveolar lavage fluid (BAL) from CF, including IL-8, IL-1, IL-6 and tumour necrosis factor alpha (TNFα) [7], leukotriene B₄ (LTB₄), complement factors, such as C5a, and bacterial products also act as potent chemoattractants for neutrophils [2]. Neutrophil elastase (NE) has also been shown to induce gene activation and secretion of IL-8 and digestion of C3b receptors on neutrophils, which limits the phagocytosis of pathogens [8]. In addition, levels of the anti-inflammatory cytokine IL-10 are decreased in BAL from CF patients [9]. These events in combination recruit more neutrophils to the lungs, which in turn recruit even more neutrophils setting up a perpetual inflammatory process, which ultimately causes irreparable lung damage in CF.

Neutrophils isolated from CF patients display a different pattern of response to inflammatory mediators to that observed from normals [10], [11]. For example, neutrophils and eosinophils isolated from CF patients have been shown to have an altered arachidonic acid turnover [12], and an increased release of myeloperoxidase (MPO), eosinophil cationic protein (ECP), and eosinophil protein X (EPX) [13]. In addition, isolated peripheral neutrophils from CF patients generated higher levels of elastase when exposed to stimuli such as IL-8 and TNFα compared to neutrophils isolated from controls [14]. The chemotactic responsiveness of neutrophils to certain cytokines is also significantly lower in CF neutrophils compared to normal neutrophils [15], [16], [17]. Studies on bacterial products, such as B. cepacia lipopolysaccharide (LPS), have shown that the inflammatory nature of the B. cepacia infection in CF patients may contribute to increased neutrophil recruitment and priming of the neutrophil respiratory burst [17]. This study uses N-formyl-methionyl-leucyl-phenylalanine (fMLP), a compound which mimics bacterial chemotaxins and acts via a cell surface receptor, and phorbol 12-myristate 13-acetate (PMA), an analogue of diacylglycerol which activates protein kinase C directly. These compounds were chosen to investigate the receptor- and nonreceptor-activated stimulation of superoxide generation and elastase release from peripheral blood neutrophils isolated from CF patients before and after an exacerbation compared to neutrophils from normal healthy age and sex matched controls.

The hypothesis for this study was that neutrophil activity, as evidenced by the spontaneous and stimulated release of neutrophil elastase and superoxide production, would be greater in CF patients before resolution of the exacerbation. Therefore, the aim of the present study was to elucidate the effects of cystic fibrosis exacerbations on the reactivity of peripheral blood neutrophils using fMLP and PMA to investigate the rate of stimulated superoxide generation and elastase release compared to neutrophils from a normal healthy control population.

Section snippets

Subjects

Patients were recruited from those admitted to the adult CF unit of the Belfast City Hospital for a pulmonary exacerbation defined as: increased purulent sputum, decrease in FEV₁ of 10% or greater from previous best, weight loss, and decreased energy. Patients also had a documented sweat test and/or genetic analysis confirming the CF diagnosis. Patients were studied before and after resolution of the CF exacerbation. CF patients were treated with antibiotics for a minimum of 2 weeks until lung

Characterisation of the subjects

Patient and control characteristics are shown in Table 1. A total of 12 subjects was recruited in each group and the mean age of the subjects did not differ significantly. Four CF patients were colonised with B. cenocepacia. CF patients were treated with antibiotics for a minimum of 2 weeks until lung function returned to normal for CF. Before and after treatment, FEV₁ and FVC were significantly lower in the CF patient group compared to normal controls. As expected, FEV₁ and FVC were

Discussion

This is the first study to investigate the effects of cystic fibrosis exacerbations on peripheral blood neutrophil function. The aim of the present study was to investigate the effects of cystic fibrosis exacerbations on the reactivity of peripheral blood neutrophils using fMLP-, and PMA-stimulated superoxide generation and elastase release and compare these data to a normal healthy, age- and sex-matched control population. These stimulants were chosen because they are potent activators of

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Cited by (26)

Levels of pro- and anti-inflammatory cytokines in cystic fibrosis patients with or without gingivitis
2020, Cytokine
Citation Excerpt :
Due to reduced killing and antigen presenting ability of macrophages, persistent infection occurs [7–9]. Pro-inflammatory response in lungs, caused by persistent infection of P. aeruginosa, leads to lung destruction [10]. During the evaluation of serum and bronchoalveolar lavage fluid, increasing of pro-inflammatory cytokines like interleukin (IL)-1β, IL-17, etc. and decreasing anti-inflammatory cytokines such as IL-10 is detected in CF patients [11–13].
Inflammatory periodontal diseases are caused by interaction between gram negative, anaerobic bacteria and host response. Persistent infection of Pseudomonas aeruginosa in cystic fibrosis (CF) patients also cause increased pro-inflammatory response and the imbalance of pro- and anti-inflammatory response in brochoalveolar lavage fluid which leads to destruction of lungs. The aim of this study is to evaluate periodontal status of CF patients, to measure level of cytokines and biochemical molecules in gingival crevicular fluid (GCF), and to detect presence of P. aeruginosa in dental plaque samples.
GCF samples were collected from 41 CF patients and 39 healthy (non-CF) subjects. Interleukin (IL)-1ß, IL-17, IL-10, human neutrophil elastase (HNE), cystic fibrosis transmembrane regulator (CFTR) protein, and human β-defensin-1 (HBD1) in GCF were evaluated by ELISA method. Dental plaque samples were collected from 18 CF patients with history of P. aeruginosa colonization and 15 non-CF subjects. Presence of P. aeruginosa was evaluated by using conventional culture methods and molecular methods.
Levels of IL-1ß, HNE, and HBD1 in CF patients were significantly higher than non-CF subjects. However, IL-10 level was significantly lower in CF patients. Increased pro-inflammatory (IL-1ß) and decreased anti-inflammatory (IL-10) cytokine levels were observed in GCF samples from CF patients, irrespective of their periodontal status. P. aeruginosa were detected in four samples of 18 CF patients, and all were negative in non-CF group.
As a result of this study, CF coexists increasing pro-inflammatory and decreasing anti-inflammatory response locally. Due to increasing pro-inflammation, CF patients should be followed-up more often than non-CF children.
Increase in interleukin-8 production from circulating neutrophils upon antibiotic therapy in cystic fibrosis patients
2012, Journal of Cystic Fibrosis
Citation Excerpt :
Nevertheless, these differences in IL-8 production are unlikely due to the apoptosis since PMN apoptosis in basal conditions was found at similar level in controls and patients (Fig. 3). In our study, while ROS production is insensitive to antibiotic treatment, as also demonstrated by a previous study [29], PMNs respond to antibiotics with a significant increase in IL-8 secretion. The levels of other pro-inflammatory cytokines showed a trend to decrease for IL-1β and a significant lower level for IL-6 after therapy, ruling out that the effect on IL-8 was unspecific.
It is not known whether antibiotic therapy for lung disease in cystic fibrosis (CF) has an influence on circulating polymorphonuclear neutrophil (PMN) function and apoptosis.
Blood PMNs were obtained from 14 CF patients before and after antibiotic treatment for an acute exacerbation, and from 10 healthy controls. PMNs were evaluated for production of reactive oxygen species (ROS) by spectrophotometry, of cytokines in the conditioned medium by ELISA, and apoptotic response by cytofluorimetry.
ROS and interleukin (IL)-8 were produced at higher levels by CF PMNs pre-therapy than control PMNs under basal conditions. IL-8 levels further increased after therapy. Early apoptotic response was higher in CF PMNs pre-therapy than in control PMNs, and this pattern did not change after antibiotic treatment.
Circulating PMNs are primed in CF acute patients. Further studies are needed to consider PMN-produced IL-8 as a biomarker to evaluate response to antibiotic therapy in CF patients.
Anti-inflammatory effects of DX-890, a human neutrophil elastase inhibitor
2012, Journal of Cystic Fibrosis
Neutrophil elastase (NE)-mediated inflammation contributes to lung damage in cystic fibrosis (CF). We investigated if DX-890, a small-protein NE inhibitor, could reduce neutrophil trans-epithelial migration and reduce activity released from neutrophils and NE-induced cytokine expression in airway epithelial cells.
Activated blood neutrophils (CF and healthy) treated ± DX-890 were assayed for NE activity. Transmigration of calcein-labeled neutrophils was studied using a 16HBE14o⁻ epithelial monolayer. IL-8 release from primary nasal epithelial monolayers (CF and healthy) was measured after treatment ± DX-890 and NE or CF sputum.
DX-890 reduced NE activity from neutrophils (CF and healthy) and reduced neutrophil transmigration. DX-890 pre-treatment reduced IL-8 release from epithelial cells of healthy or CF subjects after stimulation with NE and CF sputum sol. All improvements with DX-890 were statistically significant (p < 0.05).
DX-890 reduces NE-mediated transmigration and inflammation. NE inhibition could be useful in managing neutrophilic airway inflammation in CF.
A novel neutrophil derived inflammatory biomarker of pulmonary exacerbation in cystic fibrosis
2012, Journal of Cystic Fibrosis
Citation Excerpt :
Serum levels of S100A12 [34], S100A8/A9, CRP and vascular endothelial growth factor [35] have also been suggested as serum markers of acute infectious exacerbations and to decrease significantly post treatment of an exacerbation. Moreover, while CF exacerbations were not shown to modulate neutrophil function [36], neutrophil derived proteins including myeloperoxidase in peripheral blood appeared to reflect inflammatory changes post antibiotic treatment [37]. Myeloperoxidase however is a component of neutrophil primary/azurophilic granules and peroxidase exocytosis is a tightly regulated process.
The focus of this study was to characterize a novel biomarker for cystic fibrosis (CF) that could reflect exacerbations of the disease and could be useful for therapeutic stratification of patients, or for testing of potential drug treatments. This study focused exclusively on a protein complex containing alpha-1 antitrypsin and CD16b (AAT:CD16b) which is released into the bloodstream from membranes of pro-inflammatory primed neutrophils.
Neutrophil membrane expression and extracellular levels of AAT and CD16b were quantified by flow cytometry, Western blot analysis and by 2D-PAGE. Interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and AAT:CD16b complex were quantified in CF plasma (n = 38), samples post antibiotic treatment for 14 days (n = 10), chronic obstructive pulmonary disease (n = 10), AAT deficient (n = 10) and healthy control (n = 14) plasma samples by ELISA.
Cell priming with IL-8 and TNF-alpha caused release of the AAT:CD16b complex from the neutrophil cell membrane. Circulating plasma levels of IL-8, TNF-alpha and AAT:CD16b complex were significantly higher in patients with CF than in the other patient groups or healthy controls (P < 0.05). Antibiotic treatment of pulmonary exacerbation in patients with CF led to decreased plasma protein concentrations of AAT:CD16b complex with a significant correlation with improved FEV1 (r = 0.81, P = 0.003).
The results of this study have shown that levels of AAT:CD16b complex present in plasma correlate to the inflammatory status of patients. The AAT:CD16b biomarker may become a useful addition to the clinical diagnosis of exacerbations in CF.
Evidence of neutrophil functional defect despite inflammation in stable cirrhosis
2011, Journal of Hepatology
Citation Excerpt :
Another intriguing analogy with the cystic fibrosis model is the finding of high elastase levels in the blisters induced in cirrhotic patients which is consistent with the previous description of high circulating level of neutrophil elastase in cirrhosis [25] and with the key role of this enzyme in the pathophysiology of cystic fibrosis [26]. Neutrophils from patients with cystic fibrosis spontaneously release excess elastase [27] thus contributing to the damage to the cystic fibrosis airway [28]. Furthermore, neutrophil elastase impairs neutrophil phagocytic capacity through the cleavage of the opsonin receptor CR1 and the chemokine receptor CXCR1 on neutrophil surface [29,30].
Deranged neutrophil function in alcoholic hepatitis has been shown to be transmissible to normal neutrophils by patient plasma. The aims of this study were (i) to evaluate whether patients with stable cirrhosis have a similar transmissible neutrophil defect and (ii) to explore the possible mechanisms.
Plasma samples from 108 stable cirrhotic patients (Child A or B: 58; Child C: 50) and matched controls were incubated with normal neutrophils. Neutrophil resting respiratory burst, phagocytosis, and toll-like receptors 2, 4, and 9 expressions as well as plasma endotoxin, bacterial DNA, and cytokines were measured. In a separate study, eight patients and five controls were studied using a novel ‘skin-window’ technique to evaluate neutrophil function in an area of induced sterile inflammation.
Patient plasma induced neutrophil phagocytic dysfunction was greater in patients with more severe disease and was associated with increased expression of toll-like receptors 2 and 4. An increased resting respiratory burst was observed in a subset of patients, showing higher levels of inflammatory cytokines and more pronounced phagocytic impairment. No correlation was found with endotoxemia or bacterial DNA. In patients with compensated cirrhosis and apparently normal neutrophil function, the ‘skin-window’ study disclosed a severe phagocytic defect at the site of inflammation. Significantly higher levels of neutrophil elastase and IL-8 were found in the blister fluid.
Stable cirrhosis is characterized by neutrophil phagocytic dysfunction which may be subtle and only revealed in inflamed peripheral tissues where excessive inflammatory mediators continue to be released.
Inactivation of IL-6 and soluble IL-6 receptor by neutrophil derived serine proteases in cystic fibrosis
2010, Biochimica et Biophysica Acta - Molecular Basis of Disease
The ability of IL-6 to signal via both membrane bound and soluble receptors is thought to explain the capacity of this cytokine to act in both the initiation and resolution of acute inflammatory responses. In cystic fibrosis (CF), poorly resolved neutrophillic inflammation of the lungs is a primary cause of morbidity and mortality. Expression of IL-6 has been reported to be low in CF lung secretions, despite ongoing inflammation, but the status of soluble IL-6 receptor (sIL-6R) in these patients is unknown. We hypothesised that sIL-6R may be an important potentiator of IL-6 activity in CF associated lung disease. IL-6, sIL-6R and sgp130 (a natural antagonist of responses mediated by the sIL-6R) were analysed by ELISA and Western blot in bronchoalveolar lavage fluid (BALF) from 28 paediatric CF patients and nine non-CF controls. Total cell counts in CF were four fold higher compared to controls (median: 1.4 × 10⁶ cells/ml v. 0.35 × 10⁶ cells/ml in controls) (p < 0.001) and the infiltrate was dominated by neutrophils which were elevated by 89 fold (0.62 × 10⁶ cells/ml v. 0.007 × 10⁶ cells/ml in controls) (p < 0.001). Other markers of inflammation such as IL-8 and MCP-1 were elevated 17.5 and 3.8 fold respectively (IL-8; median: 1122 pg/ml v. 64 pg/ml in controls, p < 0.01 and MCP-1; median: 692 pg/ml v. 182 pg/ml in controls, p < 0.05). IL-6, although present in 23/32 CF BALF specimens compared to 1/9 controls (p < 0.01), was weakly expressed (median: 50 pg/ml). Expression of sIL-6R and sgp130 in CF was no different to control patients. We tested whether weak expression of all three molecules was due to degradation by CF BALF. Degradative activity was observed in association with BALF elastase activity and could be specifically blocked by serine protease inhibitors. Degradation of sIL-6R by purified serine proteases (elastase, cathepsin G and proteinase 3) was also observed leading to a loss of trans-signalling activity. Interestingly, sIL-6R was protected from proteolysis by interaction with IL-6. Our data identify and define a novel protease mediated deficiency of IL-6 signalling in the CF lung.

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Effect of cystic fibrosis exacerbations on neutrophil function

Abstract

Introduction

Section snippets

Subjects

Characterisation of the subjects

Discussion

Blood

Biochim. Biophys. Acta

Trends Biomed. Sci.

Immunological aspects of lung diseases and cystic fibrosis

JAMA

Current understanding of the inflammatory process in cystic fibrosis: onset and etiology

Pediatr. Pulmonol.

Burkholderia cepacia: medical, taxonomic and ecological issues

J. Med. Microbiol.

Cystic fibrosis and pulmonary involvement from multiple perspectives

Semin. Respir. Infect.

Bronchoalveolar lavage findings in cystic inflammation

Am. J. Respir. Crit. Care Med.

Tissue injury in neutrophil inflammation

Inflamm. Res.

Normal bronchial epithelial cells constitutively produce the anti-inflammatory cytokine IL-10 which is down regulated in cystic fibrosis

Am. J. Respir. Cell Mol. Biol.

Elastases and emphysema: current assessment of the protease–antiprotease hypothesis

Am. Rev. Respir. Dis.

Inflammatory cytokines in cystic fibrosis lungs

Am. J. Respir. Crit. Care Med.

Neutrophil adhesion molecule surface expression and responsiveness in cystic fibrosis

Am. J. Respir. Crit. Care Med.

Priming of blood neutrophils in children with cystic fibrosis: correlation between function and phenotypic expression of opsonin receptors before and after platelet-activating factor priming

J. Infect. Dis.