ABSTRACT
The collectin surfactant protein D (SP-D) is an important component of the pulmonary innate host defence. Up to now, little is known about the regulation of eosinophil function by SP-D. Various murine models of pulmonary hypersensitivity suggest that SP-D may be a potent anti-allergic protein. We investigated the modulation of eosinophil chemotaxis and degranulation by human SP-D. SP-D markedly inhibited the chemotaxis of eosinophils triggered by eotaxin, a major tissue-derived CC-chemokine, as shown in a modified Boyden chamber assay. In addition, degranulation of ECP in response to Ca2+ ionophore, immobilized IgG and serum from allergic patients was inhibited by SP-D. In a fixed-cell enzyme linked immunosorbent assay and in flow cytometry, SP-D bound to eosinophils. This binding was saturable and was inhibited by the addition of maltose and ethylenediaminetetraacetic acid, suggesting the involvement of the carbohydrate recognition domain of SP-D. In addition, flow cytometry showed significant interaction of SP-D with CD32 (FcgammaII receptor) on eosinophils, which might explain the inhibitory effect of SP-D on the IgG and serum-triggered eosinophil cationic protein degranulation of eosinophils. Our data further support the concept of an anti-inflammatory function of SP-D in the lung of patients with allergic diseases.
Subject(s)
Eosinophils/metabolism , Pulmonary Surfactant-Associated Protein D/pharmacology , Adult , Analysis of Variance , Asthma/metabolism , Bronchoalveolar Lavage Fluid/chemistry , Cell Degranulation/drug effects , Cells, Cultured , Chemokine CCL11 , Chemokines, CC , Chemotaxis/drug effects , Electrophoresis, Polyacrylamide Gel , Eosinophil Cationic Protein/metabolism , Eosinophils/drug effects , Female , Flow Cytometry , Humans , Male , Protein Binding/drug effects , Pulmonary Alveolar Proteinosis/metabolism , Pulmonary Surfactant-Associated Protein D/isolation & purification , Pulmonary Surfactant-Associated Protein D/metabolism , Receptors, IgG/metabolismABSTRACT
Severe respiratory infections are a major cause of morbidity and mortality in children receiving immunosuppressive therapy for malignancies. The goal of this study was to assess the major changes in the protein patterns in these children. Bronchoalveolar lavage (BAL) fluids of seven control children and of ten children with malignancies and fever not responding to broad spectrum antibiotic treatment was separated by horizontal two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the isoelectric point range 3-9. We observed a large increase of alpha(1)-antitrypsin (p = 0.0004) and decreases of the immunoglobulin (Ig) binding factor, transthyretin and cystatin S. Significant changes occurred also in the small acidic proteins. The relative abundance of the IgG heavy and light chains may hinder the separation and identification of many minor protein spots located in the basic area of the gel, suggesting that their removal during sample preparation may be warranted. This study demonstrated significant alterations in BAL fluid proteome in immunosuppressed children with persistent fever and pulmonary infiltrates. Future target regions of interest were identified. Sample prefractionation and the selection of suitable narrow isoelectric point ranges will be necessary for optimized detection and separating conditions.
Subject(s)
Bronchoalveolar Lavage Fluid , Electrophoresis, Gel, Two-Dimensional/methods , Lung Diseases/metabolism , Neoplasms/metabolism , Adolescent , Anemia/metabolism , Child , Child, Preschool , Female , Humans , Immunosuppression Therapy , Leukemia/metabolism , Male , Pilot ProjectsABSTRACT
The proteins recovered in exhaled breath condensate (EBC) might be used to non-invasively monitor respiratory diseases. However, the range of proteins and their source are still unresolved and contamination by saliva or a similar protein pattern in the nasal and bronchial compartments may make interpretation of the data difficult. We studied nasal EBC (collected through a "free of touch" technique by negative pressure), oral tidal, and oral forced EBC (collected through a rebreathing valve as a saliva trap connected to tubing submerged into ice) and matched saliva samples from five healthy adult subjects. The protein samples were separated by two-dimensional electrophoresis and the silver stained gels were analyzed by Melanie 2 software. In both nasal and oral EBC, three spots (72 kDa/isoelectric point (pI) 6.6-7.0, 66 kDa/pI 5.9-6.7 and 45-48 kDa/pI 8.0-8.6) were consistently present in all subjects. Several other proteins were only sporadically detected. Despite improbable saliva contamination (no phosphorus contamination in the same oral and nasal EBC, no amylase activity in 10 pairs of nasal and oral EBC collected by the same technique), on average 63% and 71% of the spots identified in oral and nasal EBC were also found in the matched individual saliva samples. Compared to saliva, the range and amount of protein in all types of EBC was very small. Even when collected free of saliva contamination the majority of proteins present in EBC was also found in saliva, suggesting that these proteins are present in both compartments, e.g. saliva and secretions of the lower airspaces. The quantification and identification of specific proteins in the various compartments is warranted in future studies to determine the practical value of EBC.
