ABSTRACT
The transient receptor potential cation channel family member ankyrin 1 (TRPA1) is a potential target for several diseases, but detection of human TRPA1 (hTRPA1) protein in cells and tissues is problematic as rigorous antibody validation is lacking. We expressed hTRPA1 in a TRPA1-negative cell line to evaluate 5 commercially available antibodies by western blotting, immunofluorescence, immunocytochemistry and flow cytometry. The three most cited anti-TRPA1 antibodies lacked sensitivity and/or specificity, but two mouse monoclonal anti-TRPA1 antibodies detected hTRPA1 specifically in the above assays. This enabled the development of a flow cytometry assay, which demonstrated strong expression of TRPA1 in human lung myofibroblasts, human airway smooth muscle cells but not lung mast cells. The most cited anti-TRPA1 antibodies lack sensitivity and/or specificity for hTRPA1. We have identified two anti-TRPA1 antibodies which detect hTRPA1 specifically. Previously published data regarding human TRPA1 protein expression may need revisiting.
Subject(s)
Antibodies/chemistry , Myocytes, Smooth Muscle/metabolism , Myofibroblasts/metabolism , TRPA1 Cation Channel/metabolism , Calcium/metabolism , Calcium Signaling , Epitopes/chemistry , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Immunohistochemistry , Lung/cytology , Microscopy, FluorescenceABSTRACT
BACKGROUND: The role of fibrocytes in chronic obstructive pulmonary disease (COPD) is unknown. We sought to enumerate blood and tissue fibrocytes in COPD and determine the association of blood fibrocytes with clinical features of disease. METHODS: Utilizing flow cytometry to identify circulating, collagen type 1+ cells, we found two populations: (i) CD45+ CD34+ (fibrocytes) and (ii) CD45+ CD34- [myeloid-derived suppressor cell (MDSC)-like fibrocytes] cells in stable COPD (n = 41) and control (n = 29) subjects. Lung resection material from a separate group of subjects with (n = 11) or without (n = 11) COPD was collected for tissue fibrocyte detection. We examined circulating fibrocyte populations for correlations with clinical parameters including quantitative computed tomography (qCT) and determined pathways of association between correlated variables using a path analysis model. RESULTS: Blood and tissue fibrocytes were not increased compared to control subjects nor were blood fibrocytes associated with lung function or qCT, but were increased in eosinophilic COPD. Myeloid-derived suppressor cell-like fibrocytes were increased in COPD compared to controls [2.3 (1.1-4.9), P = 0.038]. Our path analysis model showed that collagen type 1 intensity for MDSC-like fibrocytes was positively associated with lung function through associations with air trapping, predominately in the upper lobes. CONCLUSION: We have demonstrated that two circulating populations of fibrocyte exist in COPD, with distinct clinical associations, but are not prevalent in proximal or small airway tissue. Blood MDSC-like fibrocytes, however, are increased and associated with preserved lung function through a small airway-dependent mechanism in COPD.
Subject(s)
Fibroblasts/pathology , Myeloid-Derived Suppressor Cells/pathology , Pulmonary Disease, Chronic Obstructive/pathology , Pulmonary Disease, Chronic Obstructive/physiopathology , Aged , Biomarkers , Case-Control Studies , Cell Count , Cell Differentiation , Female , Fibroblasts/metabolism , Flow Cytometry , Humans , Immunophenotyping , Male , Middle Aged , Myeloid-Derived Suppressor Cells/metabolism , Phenotype , Pulmonary Disease, Chronic Obstructive/blood , Pulmonary Disease, Chronic Obstructive/diagnosis , Respiratory Function Tests , Severity of Illness Index , Tomography, X-Ray ComputedABSTRACT
Mucosal vaccination against pneumococcal disease offers potential protection against otitis media, pneumonia and invasive disease, including providing herd benefit by reducing pathogen carriage. The major obstacle, however, remains the lack of a suitable adjuvant for use in humans. Animal models have demonstrated success of interleukin-12 (IL-12) as an adjuvant for mucosal vaccines using recombinant pneumococcal protein antigens. This review examines the biology of the IL-12 cytokine family, the toxicity of IL-12 in human studies and suggests approaches by which IL-12 could be developed as a mucosal adjuvant with pneumococcal protein based vaccines, for use in humans.
