GM-CSF gene targeted (GM(-/-)) mice are susceptible to respiratory infections and develop alveolar proteinosis due to defects in innate immune function and surfactant catabolism in alveolar macrophages (AMs), respectively. Reduced cell adhesion, phagocytosis, pathogen killing, mannose- and Toll-like receptor expression, and LPS- or peptidoglycan-stimulated TNFalpha release were observed in AMs from GM(-/-) mice. The transcription factor PU.1 was markedly reduced in AMs of GM(-/-) mice in vivo and was restored by selective expression of GM-CSF in the lungs of SPC-GM/GM(-/-) transgenic mice. Retrovirus-mediated expression of PU.1 in AMs from GM(-/-) mice rescued host defense functions and surfactant catabolism by AMs. We conclude that PU.1 mediates GM-CSF-dependent effects on terminal differentiation of AMs regulating innate immune functions and surfactant catabolism by AMs.

LiteBIRD is a next-generation satellite mission to measure the polarization of the cosmic microwave background (CMB) radiation. On large angular scales the B-mode polarization of the CMB carries the imprint of primordial gravitational waves, and its precise measurement would provide a powerful probe of the epoch of inflation. The goal of LiteBIRD is to achieve a measurement of the characterizing tensor to scalar ratio r to an uncertainty of δr = 0.001. In order to achieve this goal we will employ a kilopixel superconducting detector array on a cryogenically cooled sub-Kelvin focal plane with an optical system at a temperature of 4 K. We are currently considering two detector array options; transition edge sensor (TES) bolometers and microwave kinetic inductance detectors (MKID). In this paper we give an overview of LiteBIRD and describe a TES-based polarimeter designed to achieve the target sensitivity of 2 µK·arcmin over the frequency range 50 to 320 GHz.

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Targeted ablation of the surfactant protein D (SP-D) gene caused chronic inflammation, emphysema, and fibrosis in the lungs of SP-D (؊͞؊) mice. Although lung morphology was unperturbed during the first 2 weeks of life, airspace enlargement was observed by 3 weeks and progressed with advancing age. Inflammation consisted of hypertrophic alveolar macrophages and peribronchiolar-perivascular monocytic infiltrates. These abnormalities were associated with increased activity of the matrix metalloproteinases, MMP2 and MMP9, and immunostaining for MMP9 and MMP12 in alveolar macrophages. Hydrogen peroxide production by isolated alveolar macrophages also was increased significantly (10-fold). SP-D plays a critical role in the suppression of alveolar macrophage activation, which may contribute to the pathogenesis of chronic inflammation and emphysema.

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Tetraspanins CD9 and CD81 facilitate the fusion between gametes, myoblasts, or virus-infected cells. Here, we investigated the role of these tetraspanins in the fusion of mononuclear phagocytes. Expression of CD9 and CD81 and their complex formation with integrins were up-regulated when blood monocytes were cultured under normal conditions. Under fusogenic conditions in the presence of Con A, CD9 and CD81 up-regulation was inhibited, and their complex formation with integrins was down-regulated. Anti-CD9 and -CD81 antibodies, which were previously shown to inhibit the fusion of gametes, myoblasts, and virus-infected cells, unexpectedly promoted the fusion of monocytes and alveolar macrophages. However, these effects were not due to altered cell adhesion, aggregation, or cytokine production. When stimulated in vitro or in vivo, alveolar macrophages and bone marrow cells of CD9- and CD81-null mice formed larger numbers of multinucleated cells than those of wild-type mice. Finally, CD9/CD81 double-null mice spontaneously developed multinucleated giant cells in the lung and showed enhanced osteoclastogenesis in the bone. These results suggest that CD9 and CD81 coordinately prevent the fusion of mononuclear phagocytes.

Acute lung injury syndromes remain common causes of morbidity and mortality in adults and children. Cellular and physiologic mechanisms maintaining pulmonary homeostasis during lung injury remain poorly understood. In the present study, the Stat-3 gene was selectively deleted in respiratory epithelial cells by conditional expression of Cre-recombinase under control of the surfactant protein C gene promoter. Cell-selective deletion of Stat-3 in respiratory epithelial cells did not alter prenatal lung morphogenesis or postnatal lung function. However, exposure of adult Stat-3-deleted mice to 95% oxygen caused a more rapidly progressive lung injury associated with alveolar capillary leak and acute respiratory distress. Epithelial cell injury and inflammatory responses were increased in the Stat-3-deleted mice. Surfactant proteins and lipids were decreased or absent in alveolar lavage material. Intratracheal treatment with exogenous surfactant protein B improved survival and lung histology in Stat-3-deleted mice during hyperoxia. Expression of Stat-3 in respiratory epithelial cells is not required for lung formation, but plays a critical role in maintenance of surfactant homeostasis and lung function during oxygen injury. 29Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) remain a common cause of morbidity and mortality in both infants and adults following infection, trauma, inhalation of toxicants, and drowning. The recent severe acute respiratory syndrome (SARS) outbreak emphasizes the severity of pulmonary outcomes associated with ALI (7). ARDS is associated with surfactant deficiency and dysfunction (8, 9), including abnormalities in surfactant lipids and proteins. Of the surfactant proteins, SP-B is required for maintenance of lung function in both newborn and postnatal periods (10, 11) and for adaptation to lung injury following infection or oxygen exposure. To assess the role of STAT-3 in lung function, we used a conditional system to express Cre-recombinase to selectively delete the Stat-3 gene in bronchiolar and alveolar epithelial cells of the mouse lung. MethodsGene construction and PCR. We generated SP-C-rtTA tg/-transgenic mice and (tetO) 7 CMV-Cre tg/tg or (tetO) 7 CMVCre tg/-mice as described previously (12, 13 -C-rtTA, (tetO) 7 CMV-Cre and Stat-3 flx genes using the primers 5′-GAC ACA TAT AAG ACC CTG GCTA-3′ and 5′-AAA ATC TTG CCA GCT TTC CC-3′ for SP-C-rtTA; 5′-TGC CAC CAA GTG ACA GCA ATG-3′ and 5′-AGA GAC GGA AAT CCA TCG CTCG-3′ for (tetO) 7 CMV-Cre; and 5′-CCT GAA GAC CAA GTT CAT CTG TGT GAC-3′ and 5′-CAC ACA AGC CAT CAA ACT CTG GTC TCC-3′ for Stat-3 flx . Triple transgenic (Stat-3 ∆/∆ ) and nondeleted littermate (Stat-3 flx/flx ) control mice were used for the experiments.Animal use and doxycycline administration. Transgenic Stat-3 ∆/∆ and control mice were kept in a pathogen-free vivarium according to institutional guidelines until oxygen exposure. Doxycycline was administered to the dams in the food at a concentration of 625 mg/kg (Harlan Teklad, Madison, Wisconsin, U...

Targeted ablation of the surfactant protein D (SP-D) gene caused progressive pulmonary emphysema associated with pulmonary infiltration by foamy alveolar macrophages (AMs), increased hydrogen peroxide production, and matrix metalloproteinase (MMP)-2, -9, and -12 expression. In the present study, the mechanisms by which SP-D influences macrophage MMP activity were assessed in AMs from SP-D−/− mice. Tissue lipid peroxides and reactive carbonyls were increased in lungs of SP-D−/− mice, indicating oxidative stress. Immunohistochemical staining of AMs from SP-D−/− mice demonstrated that NF-κB was highly expressed and translocated to the nucleus. Increased NF-κB binding was detected by EMSA in nuclear extracts of AMs isolated from SP-D−/− mice. Antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate inhibited MMP production by AMs from SP-D−/− mice. To assess whether increased oxidant production influenced NF-κB activation and production of MMP-2 and -9, AMs from SP-D−/− mice were treated with the NADPH oxidase inhibitors diphenylene iodonium chloride and apocynin. Inhibition of NADPH oxidase suppressed NF-κB binding by nuclear extracts and decreased production of MMP-2 and 9 in AMs from SP-D−/− mice. SN-50, a synthetic NF-κB-inhibitory peptide, decreased MMP production by AMs from SP-D−/− mice. Oxidant production and reactive oxygen species were increased in lungs of SP-D−/− mice, in turn activating NF-κB and MMP expression. SP-D plays an unexpected inhibitory role in the regulation of NF-κB in AMs.