Rationale: Lung fibroblasts are key mediators of fibrosis resulting in accumulation of excessive interstitial collagen and extracellular matrix, but their origins are not well defined. Objectives: We aimed to elucidate the contribution of lung epithelium-derived fibroblasts via epithelial-mesenchymal transition (EMT) in the intratracheal bleomycin model. Methods: Primary type II alveolar epithelial cells were cultured from Immortomice and exposed to transforming growth factor-b 1 and epidermal growth factor. Cell fate reporter mice that permanently mark cells of lung epithelial lineage with b-galactosidase were developed to study EMT, and bone marrow chimeras expressing green fluorescent protein under the control of the fibroblast-associated S100A4 promoter were generated to examine bone marrow-derived fibroblasts. Mice were given intratracheal bleomycin (0.08 unit). Immunostaining was performed for S100A4, b-galactosidase, green fluorescent protein, and a-smooth muscle actin. Measurements and Main Results: In vitro, primary type II alveolar epithelial cells undergo phenotypic changes of EMT when exposed to transforming growth factor-b 1 and epidermal growth factor with loss of prosurfactant protein C and E-cadherin and gain of S100A4 and type I procollagen. In vivo, using cell fate reporter mice, approximately one-third of S100A4-positive fibroblasts were derived from lung epithelium 2 weeks after bleomycin administration. From bone marrow chimera studies, one-fifth of S100A4-positive fibroblasts were derived from bone marrow at this same time point. Myofibroblasts rarely derived from EMT or bone marrow progenitors. Conclusions: Both EMT and bone marrow progenitors contribute to S100A4-positive fibroblasts in bleomycin-induced lung fibrosis. However, neither origin is a principal contributor to lung myofibroblasts.

apoptosis ͉ cancer ͉ inflammation ͉ airway ͉ adenocarcinoma

Airway mucus is a hallmark of respiratory syncytial virus (RSV) lower respiratory tract illness. Laboratory RSV strains differentially induce airway mucus production in mice. Here, we tested the hypothesis that RSV strains differ in pathogenesis by screening six low-passage RSV clinical isolates for mucogenicity and virulence in BALB/cJ mice. The RSV clinical isolates induced variable disease severity, lung interleukin-13 (IL-13) levels, and gob-5 levels in BALB/cJ mice. We chose two of these clinical isolates for further study. Infection of BALB/cJ mice with RSV A2001/2-20 (2-20) resulted in greater disease severity, higher lung IL-13 levels, and higher lung gob-5 levels than infection with RSV strains A2, line 19, Long, and A2001/3-12 (3-12). Like the line 19 RSV strain, the 2-20 clinical isolate induced airway mucin expression in BALB/cJ mice. The 2-20 and 3-12 RSV clinical isolates had higher lung viral loads than laboratory RSV strains at 1 day postinfection (p.i.). This increased viral load correlated with higher viral antigen levels in the bronchiolar epithelium and greater histopathologic changes at 1 day p.i. The A2 RSV strain had the highest peak viral load at day 4 p.i. RSV 2-20 infection caused epithelial desquamation, bronchiolitis, airway hyperresponsiveness, and increased breathing effort in BALB/cJ mice. We found that RSV clinical isolates induce variable pathogenesis in mice, and we established a mouse model of clinical isolate strain-dependent RSV pathogenesis that recapitulates key features of RSV disease.Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and viral pneumonia in children. Each year in the United States, RSV causes lower respiratory tract illness (LRI) in 20 to 30% of infants and leads to the hospitalization of approximately 1% of infants at a cost of $300 to $400 million (19,21,27). The incidence and disease severity of RSV can vary from year to year (47). Dominant circulating RSV strains are generally replaced each year, likely by a process involving immune selection (5,6,53,54). RSV strain differences may contribute to year-to-year and/or patient-topatient variations in clinical severity.In BALB/cJ mice, laboratory RSV strains (A2, Long, and line 19) differ in their ability to cause pulmonary interleukin-13 (IL-13) and mucin expression (34, 41). We are interested in RSV-induced mucin expression in mice because mucus overabundance contributes to airway obstruction in severe RSV disease in children (2,33,44,56). IL-13 is a cytokine linked to mucus production (71). The line 19 RSV strain induces lung IL-13 and airway mucin expression in BALB/cJ mice, whereas the A2 and Long RSV strains do not (34, 41). However, the in vitro passage histories of RSV strains A2, Long, and line 19 are not defined and involve many serial passages. Thus, it is possible that mutations in these RSV laboratory strains determine pathogenesis phenotypes in the mouse model. RSV clinical isolates have not been studied extensively in vivo, and the role of RSV strain differences in...

Although the antibody-based recognition of cell-surface markers has been widely used for the identification of immune cells, overlap in the expression of markers by different cell types and the inconsistent use of antibody panels have resulted in a lack of clearly defined signatures for myeloid cell subsets. We developed a 10-fluorochrome flow cytometry panel for the identification and quantitation of myeloid cells in the lungs, including pulmonary monocytes, myeloid dendritic cells, alveolar and interstitial macrophages, and neutrophils. After the initial sorting of viable CD451 leukocytes, we detected three leukocyte subpopulations based on CD68 expression: CD68 /CD11b1 /Gr1 hi ). The validity of cellular signatures was confirmed by a morphological analysis of FACS-sorted cells, functional studies, and the depletion of specific macrophage subpopulations using liposomal clodronate. We believe our approach provides an accurate and reproducible method for the isolation, quantification, and characterization of myeloid cell subsets in the lungs, which may be useful for studying the roles of myeloid cells during various pathological processes.Keywords: interstitial macrophages; alveolar macrophages; monocytes; myeloid dendritic cells; neutrophilsThe heterogeneous population of myeloid cells in the lungs is important for maintaining homeostasis and regulating inflammation, injury, and remodeling. These cells are thought to originate from bone marrow-derived precursors, and they differentiate into mature cells with a variety of functional properties, depending on environmental cues. Multiple studies have demonstrated functional and phenotypic differences between myeloid cell subsets, including monocytes, myeloid dendritic cells, macrophages, and neutrophils. However, the specific identification and quantification of subsets of these cells in tissues are complex, and a comprehensive scheme for isolating these cells from the lungs is not readily available. To date, studies of lung myeloid cells have been limited by the inconsistent use of cellular markers and a lack of clearly defined signatures for these cells, resulting in difficulties comparing studies from different laboratories and generalizing findings across different model systems. Therefore, we undertook a study to identify and quantify myeloid cell populations comprehensively in the lungs, using flow cytometry.Based on the recent explosion of information regarding the mechanisms that underpin innate immune responses, a clear need is apparent for better strategies to identify and study myeloid cell subpopulations in the lungs. The traditional method for the identification and quantification of lung myeloid cells involves the manual counting of cells obtained from the airways by bronchoalveolar lavage (BAL). This methodology normally yields a predominance of alveolar macrophages, because these are the primary immune cells in the airspaces. Interstitial macrophages, however, comprise a substantial portion of the lung myeloid cell population (1), and may have dif...

Activation of innate immunity in the lungs can lead to a self-limited inflammatory response or progress to severe lung injury. We investigated whether specific parameters of NF-κB pathway activation determine the outcome of acute lung inflammation using a novel line of transgenic reporter mice. Following a single i.p. injection of Escherichia coli LPS, transient NF-κB activation was identified in a variety of lung cell types, and neutrophilic inflammation resolved without substantial tissue injury. However, administration of LPS over 24 h by osmotic pump (LPS pump) implanted into the peritoneum resulted in sustained, widespread NF-κB activation and neutrophilic inflammation that culminated in lung injury at 48 h. To determine whether intervention in the NF-κB pathway could prevent progression to lung injury in the LPS pump model, we administered a specific IκB kinase inhibitor (BMS-345541) to down-regulate NF-κB activation following the onset of inflammation. Treatment with BMS-345541 beginning at 20 h after osmotic pump implantation reduced lung NF-κB activation, concentration of KC and MIP-2 in lung lavage, neutrophil influx, and lung edema measured at 48 h. Therefore, sustained NF-κB activation correlates with severity of lung injury, and interdiction in the NF-κB pathway is beneficial even after the onset of lung inflammation.