ABSTRACT
Airway remodeling is strongly correlated with the progression of chronic obstructive pulmonary disease (COPD). In this study, our goal was to characterize progressive structural changes in site-specific airways, along with the temporal and spatial expression of transforming growth factor (TGF)-ß in the lungs of male spontaneously hypertensive rats exposed to tobacco smoke (TS). Our studies demonstrated that TS-induced changes of the airways is dependent on airway generation and exposure duration for proximal, midlevel, and distal airways. Stratified squamous epithelial cell metaplasia was evident in the most proximal airways after 4 and 12 weeks but with minimal levels of TGF-ß-positive epithelial cells after only 4 weeks of exposure. In contrast, epithelial cells in midlevel and distal airways were strongly TGF-ß positive at both 4 and 12 weeks of TS exposure. Airway smooth muscle volume increased significantly at 4 and 12 weeks in midlevel airways. Immunohistochemistry of TGF-ß was also found to be significantly increased at 4 and 12 weeks in lymphoid tissues and alveolar macrophages. ELISA of whole-lung homogenate demonstrated that TGF-ß2 was increased after 4 and 12 weeks of TS exposure, whereas TGF-ß1 was decreased at 12 weeks of TS exposure. Airway levels of messenger RNA for TGF-ß2, as well as platelet-derived growth factor-A, granulocyte-macrophage colony-stimulating factor, and vascular endothelial growth factor-α, growth factors regulated by TGF-ß, were significantly decreased in animals after 12 weeks of TS exposure. Our data indicate that TS increases TGF-ß in epithelial and inflammatory cells in connection with airway remodeling, although the specific role of each TGF-ß isoform remains to be defined in TS-induced airway injury and disease.
Subject(s)
Airway Remodeling/genetics , Gene Expression Regulation , Lung/metabolism , Lung/pathology , Nicotiana/adverse effects , Smoking/adverse effects , Transforming Growth Factor beta/genetics , Animals , Cell Aggregation , Epithelium/pathology , Lymphocytes/pathology , Macrophages, Alveolar/metabolism , Male , Organ Specificity/genetics , Rats, Inbred SHR , Time Factors , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta2/genetics , Transforming Growth Factor beta2/metabolism , Transforming Growth Factor beta3/genetics , Transforming Growth Factor beta3/metabolismABSTRACT
BACKGROUND: Continuous exposure to tobacco smoke (TS) is a key cause of chronic obstructive pulmonary disease (COPD), a complex multifactorial disease that is difficult to model in rodents. The spontaneously hypertensive (SH) rat exhibits several COPD-associated co-morbidities such as hypertension and increased coagulation. We have investigated whether SH rats are a more appropriate animal paradigm of COPD. METHODS: SH rats were exposed to TS for 6 hours/day, 3 days/week for 14 weeks, and the lung tissues examined by immunohistochemistry. RESULTS: TS induced a CK13-positive squamous metaplasia in proximal airways, which also stained for Ki67 and p63. We hypothesise that this lesion arises by basal cell proliferation, which differentiates to a squamous cell phenotype. Differences in staining profiles for the functional markers CC10 and surfactant D, but not phospho-p38, indicated loss of ability to function appropriately as secretory cells. Within the parenchyma, there were also differences in the staining profiles for CC10 and surfactant D, indicating a possible attempt to compensate for losses in proximal airways. In human COPD sections, areas of CK13-positive squamous metaplasia showed sporadic p63 staining, suggesting that unlike the rat, this is not a basal cell-driven lesion. CONCLUSION: This study demonstrates that although proximal airway metaplasia in rat and human are both CK13+ and therefore squamous, they potentially arise by different mechanisms.
Subject(s)
Epithelial Cells/drug effects , Hypertension/complications , Lung/drug effects , Pulmonary Disease, Chronic Obstructive/etiology , Smoking/adverse effects , Animals , Cell Differentiation , Cell Proliferation , Disease Models, Animal , Epithelial Cells/metabolism , Epithelial Cells/pathology , Humans , Hypertension/metabolism , Hypertension/pathology , Immunohistochemistry , Keratin-13/metabolism , Ki-67 Antigen/metabolism , Lung/metabolism , Lung/pathology , Metaplasia , Phenotype , Phosphorylation , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/pathology , Pulmonary Surfactant-Associated Protein D/metabolism , Rats , Rats, Inbred SHR , Species Specificity , Time Factors , Uteroglobin/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
BACKGROUND: Human lung and blood-derived mast cells express a Ca2+-activated K+ channel (KCA) that has electrophysiological properties resembling the intermediate conductance KCA (iKCA1). This channel is predicted to enhance IgE-dependent mast cell responses. OBJECTIVE: To confirm the identity of this channel as iKCA1 in human lung mast cells and to examine the effect of an iKCA1 opener, 1-ethyl-2-benzimidazolinone (1-EBIO), on Ca2+ influx and degranulation after IgE-dependent activation. METHODS: iKCA1 expression was examined by using RT-PCR. Ion currents were measured by using the patch clamp technique in human peripheral blood-derived mast cells, freshly isolated human lung mast cells (HLMCs), and long-term cultured HLMCs (LTHLMCs). Currents were manipulated with the specific iKCA1 opener 1-EBIO and the iKCA1 blockers clotrimazole and TRAM-34. Ratiometric Ca2+ imaging was performed on single fura-2-loaded cells, and histamine release was measured by radioenzymatic assay. RESULTS: Both fresh HLMCs and LTHLMCs expressed iKCA1 mRNA. The iKCA1 opener 1-EBIO induced iKCA1 currents in 89% of human peripheral blood-derived mast cells, 12% of fresh HLMCs, and 67% of LTHLMCs, which were blocked by the iKCA1 blockers clotrimazole and TRAM-34. After cell activation with a suboptimal concentration of anti-IgE, 1-EBIO enhanced the IgE-dependent rise in cytosolic-free Ca2+ and potentiated IgE-dependent histamine release. CONCLUSION: Opening of iKCA1 enhances IgE-dependent Ca2+ influx and histamine release in HLMCs. Inhibition of iKCA1 may provide a novel approach to the treatment of mast cell-mediated disease.
Subject(s)
Calcium/metabolism , Cell Degranulation/immunology , Mast Cells/immunology , Potassium Channels/metabolism , Benzimidazoles/pharmacology , Calcium Channel Agonists/pharmacology , Calcium Channel Blockers/pharmacology , Cell Degranulation/drug effects , Cells, Cultured , Clotrimazole/pharmacology , Histamine/immunology , Humans , Immunoglobulin E/immunology , Intermediate-Conductance Calcium-Activated Potassium Channels , Lung/cytology , Lung/immunology , Mast Cells/drug effects , Patch-Clamp Techniques , Potassium Channels/drug effects , Pyrazoles/pharmacologyABSTRACT
Eosinophil recruitment to airway tissue is a key feature of asthma, and release of a wide variety of toxic mediators from eosinophils leads to the tissue damage that is a hallmark of asthma pathology. Factors that control the release of these toxic mediators are targets for potential therapeutic intervention. Protease-activated receptors (PARs) are a novel class of receptors that are activated by cleavage of the N terminus of the receptor by proteases such as thrombin or trypsin-like enzymes. To date, PAR1-4 have been identified, and there are several studies that have demonstrated the expression of PARs in airway tissue, particularly the respiratory epithelium. We have investigated whether eosinophils express PARs and if activation of these receptors will then trigger a functional response. Using a combination of reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometry analysis, we have demonstrated that eosinophils express PAR1 and PAR2. FACS analysis showed that PAR1 could be clearly detected on the surface of the cells, whereas PAR2 appeared to be primarily intracellular. Trypsin and the PAR2 agonist peptide were seen in trigger shape change, release of cysteinyl leukotrienes, and most obviously, generation of reactive oxygen species. In contrast, thrombin had no effect on eosinophil function. The PAR1 agonist peptide did have a minor effect on eosinophil function, but this was most likely down to its ability to activate PAR1 and PAR2. These results demonstrate that PAR2 is the major PAR receptor that is capable of modulating eosinophil function.