The effects of the mucolytic agent, dithioerythritol (DTE), and the temperature at which sputum processing is conducted on cellular and biochemical markers in induced sputum was assessed.Samples from healthy and atopic asthmatic subjects were treated with either DTE or phosphate-buffered saline (PBS) at 22 or 378C and compared for cell counts and concentrations of histamine, tryptase, eosinophil cationic protein (ECP), free interleukin (IL)-8, immunoglobulin (Ig)A, IL-8/IgA complexes and secretory component (SC). In addition, the influence of DTE on in vitro mediator release from blood eosinophils, basophils and bronchoalveolar lavage (BAL) mast cells was studied.Processing with DTE improved cytospin quality and increased the cell yield and measurable ECP, tryptase, IgA and SC, but reduced levels of histamine in PBStreated samples and had no effect on IL-8. Cell counts or mediator levels were similar when sputum was processed at 22 or 378C, even though DTE induced blood basophils and BAL mast cells to release histamine at 378C. In spiking experiments, recovery of added ECP, tryptase, total IL-8 and histamine from sputum was similar in DTE-and PBS-processed sputum, but reduced for free IL-8 in PBS-treated samples.In conclusion, dithioerythritol improves cell and mediator recovery without causing cell activation when sputum processing is conducted at room temperature. The extent of recovery depends on the mediator studied. Eur Respir J 1999; 13: 660±667. Several studies have shown that sputum induction is safe and gives reproducible sputum cell counts and concentrations of soluble mediators [1][2][3][4][5][6]. However, the possibility that induction itself or subsequent sputum processing activates airway inflammatory cells has not been entirely ruled out [7], leaving doubt as to whether the findings in sputum accurately reflect mucosal inflammation in vivo. In particular, it remains unclear whether the use of the reducing mucolytic agents, dithioerythritol (DTE), or its optical isomer dithiothreitol (DTT), to homogenize sputum may affect the detection of mediators/proteins in the sputum fluid phase either by interfering with immunoassays or by exerting a direct effect on inflammatory cells. Furthermore, it is not known whether the temperature at which sputum is processed is important for mediator detection and cell differential counts, with some authors conducting this at room temperature [8,9] and others at 378C [4,6,[10][11][12][13].To address the above issues evidence has been sought for any effects of DTE on the detection of several inflammatory markers of relevance to airways inflammation, including differential cell counts, eosinophil cationic protein (ECP), tryptase, histamine, interleukin (IL)-8, immunoglobulin (Ig)A, and secretory component (SC). To that effect sputum samples treated with DTE or phosphatebuffered saline (PBS) were compared and the direct effects of DTE on mediator immunoassays were studied. As a further means of investigating the effect of DTE on inflammatory cells, it was inves...

These results point to an anti-inflammatory role for heparin and a novel, potentially, pro-inflammatory role for alpha2-macroglobulin which together indicate the importance of cytokine-binding macromolecules in determining net cytokine function.

The bronchial epithelium is a source of both α and β chemokines and, uniquely, of secretory component (SC), the extracellular ligand-binding domain of the polymeric IgA receptor. Ig superfamily relatives of SC, such as IgG and α2-macroglobulin, bind IL-8. Therefore, we tested the hypothesis that SC binds IL-8, modifying its activity as a neutrophil chemoattractant. Primary bronchial epithelial cells were cultured under conditions to optimize SC synthesis. The chemokines IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene α, and RANTES were released constitutively by epithelial cells from both normal and asthmatic donors and detected in high m.w. complexes with SC. There were no qualitative differences in the production of SC-chemokine complexes by epithelial cells from normal or asthmatic donors, and in all cases this was the only form of chemokine detected. SC contains 15% N-linked carbohydrate, and complete deglycosylation with peptide N-glycosidase F abolished IL-8 binding. In micro-Boyden chamber assays, no IL-8-dependent neutrophil chemotactic responses to epithelial culture supernatants could be demonstrated. SC dose-dependently (IC50 ∼0.3 nM) inhibited the neutrophil chemotactic response to rIL-8 (10 nM) in micro-Boyden chamber assays and also inhibited IL-8-mediated neutrophil transendothelial migration. SC inhibited the binding of IL-8 to nonspecific binding sites on polycarbonate filters and endothelial cell monolayers, and therefore the formation of haptotactic gradients, without effects on IL-8 binding to specific receptors on neutrophils. The data indicate that in the airways IL-8 may be solubilized and inactivated by binding to SC.

Infection of the cystic fibrosis (CF) airways elicits an exaggerated, interleukin-8 (IL-8) mediated, neutrophil inflammatory response. Necrosing neutrophils release DNA and actin into the airways, increasing the viscoelasticity of airway secretions. Mucolytics aim to improve airway clearance by reducing this viscoelasticity. DNase I reduces the viscoelasticity of CF sputum, and a human recombinant form of this enzyme is widely administered to patients with CF. Gelsolin, which cleaves actin polymers, is also known to reduce CF sputum viscosity in vitro, and it has been proposed as a future mucolytic agent. We have shown that the anionic polymers DNA and actin bind and mask immunologic recognition of the basic peptide IL-8 and prevent this chemokine from binding to neutrophil receptors. Reduction of CF sputum viscosity by DNase I or gelsolin in vitro was demonstrated to increase the proportion of free IL-8 and the IL-8-dependent neutrophil chemotactic activity of sputum supernatants. We hypothesize that an electrostatic interaction between polymer and chemokine may limit the inflammatory potential of the latter, but that this interaction may be weakened by polymer cleavage. The potential risk of increased inflammation via this mechanism suggests a caveat should be attendant on treatment of patients with CF with these mucolytic agents.

Secretory IgA contributes to humoral defense mechanisms against pathogens targeting mucosal surfaces, and secretory component (SC) fulfills multiple roles in this defense. The aims of this study were to quantify total SC and to analyze the form of free SC in sputa from normal subjects, subjects with asthma, and subjects with cystic fibrosis (CF). Significantly higher levels of SC were detected in CF compared with both other groups. Gel filtration chromatography revealed that SC in CF was relatively degraded. Free SC normally binds interleukin (IL)-8 and inhibits its function. However, in CF sputa, IL-8 binding to intact SC was reduced. Analysis of the total carbohydrate content of free SC signified overglycosylation in CF compared with normal subjects and subjects with asthma. Monosaccharide composition analysis of free SC from CF subjects revealed overfucosylation and undersialylation, in agreement with the reported CF glycosylation phenotype. SC binding to IL-8 did not interfere with the binding of IL-8 to heparin, indicating distinct binding sites on IL-8 for negative regulation of function by SC and heparin. We suggest that defective structure and function of SC contribute to the characteristic sustained inflammatory response in the CF airways.