β1 Integrin regulates adult lung alveolar epithelial cell inflammation

1Division of Neonatology, Department of Pediatrics, 2Division of Allergy, Pulmonary, and Critical Care Medicine, Department of Medicine, and 3Division of Hematology and Oncology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA. 4Nashville Veterans Affairs Medical Center, Nashville, Tennessee, USA. 5Division of Pulmonary Medicine, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. 6Division of Nephrology and Hypertension, Department of Medicine, 7Department of Molecular Physiology and Biophysics, and 8Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Alveoli are complex structures composed of epithelial cells attached to a basement membrane juxtaposed to capillaries and stromal fibroblasts. Epithelial cells are either cuboidal type 2 alveolar epithelial cells (AECs) expressing high levels of surfactant protein C (SP-C) or very thin type 1 AECs in close apposition to capillaries. We previously reported that β 1 integrin regulates branching morphogenesis and alveolarization during lung development (22). Moreover, we showed that genetically deleting β 1 integrin in the developing alveolus results in dilated airspaces, thickened alveolar septa, type 2 AEC hyperplasia, and increased numbers of alveolar macrophages. Macrophage depletion rescued the alveolarization defect in these mice (22). These findings suggest that epithelial β 1 integrin dysfunction has deleterious consequences in lung epithelium through regulation of innate immunity. The mechanisms whereby these epithelial-macrophage interactions occur are uncertain, and, perhaps more importantly, the function of β 1 integrin in the adult lung is not established.
In this study, we deleted β 1 integrin in type 2 AECs after completion of lung development, which occurs by P28. At 2 years of age, the mice developed emphysematous changes in the lung parenchyma, as Integrins, the extracellular matrix receptors that facilitate cell adhesion and migration, are necessary for organ morphogenesis; however, their role in maintaining adult tissue homeostasis is poorly understood. To define the functional importance of β 1 integrin in adult mouse lung, we deleted it after completion of development in type 2 alveolar epithelial cells (AECs). Aged β 1 integrin-deficient mice exhibited chronic obstructive pulmonary disease-like (COPD-like) pathology characterized by emphysema, lymphoid aggregates, and increased macrophage infiltration. These histopathological abnormalities were preceded by β 1 integrin-deficient AEC dysfunction such as excessive ROS production and upregulation of NF-κB-dependent chemokines, including CCL2. Genetic deletion of the CCL2 receptor, Ccr2, in mice with β 1 integrin-deficient type 2 AECs impaired recruitment of monocyte-derived macrophages and resulted in accelerated inflammation and severe premature emphysematous destruction. The lungs exhibited reduced AEC efferocytosis and excessive numbers of inflamed type 2 AECs, demonstrating the requirement for recruited monocytes/macrophages in limiting lung injury and remodeling in the setting of a chronically inflamed epithelium. These studies support a critical role for β 1 integrin in alveolar homeostasis in the adult lung.
well as lymphoid aggregates and increased macrophage accumulation, which are characteristic of patients with advanced chronic obstructive pulmonary disease (COPD). This condition was preceded by proliferation of inflamed AECs that exhibited abnormal cell-cell junctions and excessive inflammation. Reduction of monocytes and monocyte-derived macrophages caused rapid onset of emphysema in young mice, suggesting that these cells limit inflammation and injury by clearance of deranged type 2 AECs. Thus, we conclude that under physiological conditions, β 1 integrin plays a critical homeostatic role in lung epithelial cells by suppressing inflammatory signaling. β 1 Integrin-deficient type 2 AECs induce increased efferocytosis. We next performed in-depth analysis of the inflammatory status of β1 integrin-deficient mice. When we examined aged β1 rtTA mice, we noted that pro-SP-C staining often colocalized with CD68, suggesting that macrophages phagocytosed AECs in β1 rtTA but not β1 f/f lungs ( Figure 5A). This observation is consistent with efferocytosis, a tightly regulated process by which phagocytic cells ingest diseased or dying cells, thereby minimizing inflammation in the microenvironment (36)(37)(38)(39). To define the mechanisms whereby this occurred, we cultured primary type 2 AECs and measured secretion of CX3CL1, a "find me" chemokine that attracts phagocytes (40,41). We also assessed the expression levels of Cd47, whose gene product is an inhibitory "don't eat me" signal, in freshly isolated primary type 2 AECs (41,42). We found increased CX3CL1 production and reduced Cd47 mRNA expression by β1 rtTA type 2 AECs relative to type 2 AECs isolated from β1 f/f mice ( Figure 5, B and C). These findings support the conclusion that macrophage efferocytosis of β 1 integrin-deficient type 2 AECs is prominent in β1 rtTA lungs.
Deleting CCL2-recruited monocytes/macrophages causes severe destruction of alveolar architecture in β1 rtTA mice by decreasing AEC efferocytosis. Our histological examination of both aged and 3-month-old β1 rtTA lungs suggested increased inflammation. Since β1 rtTA lungs had increased macrophages, and β 1 -null type 2 AECs exhibited markers of efferocytosis, we tested whether impairment of macrophage recruitment would disrupt homeostasis in 3-month-old β1 rtTA mice. To target these recruited immune cell populations, we crossed β1 f/f and β1 rtTA mice to the Ccr2-null background. CCR2 is the receptor for CCL2, one of the primary monocyte chemokines in the lung. CCR2 -/-;β1 rtTA mice and their CCR2 -/-;β1 f/f littermate controls received dox from P28 until 2 months of age in the same manner as β1 f/f and β1 rtTA mice. In contrast to β1 rtTA mice, 3-month-old CCR2 -/-;β1 rtTA mice exhibited dramatically enhanced lung pathology ( Figure 6, A and B), with widespread emphysematous destruction; marked airspace enlargement, quantified by mean linear intercept ( Figure 6C); increased inflammatory infiltrates (arrows in Figure  6B); and increased BALF cell counts ( Figure 6D). CCR2 -/-;β1 rtTA mice exhibited a large number of CD68 + macrophages ( Figure 6, E and F) despite loss of CCL2 recruitment due to excessive proliferation of existing resident macrophages as verified by increased Ki-67 staining (Supplemental Figure 3, A-C). A large increase in pro-SP-C + type 2 AECs accompanied the expanded immune cell population ( Figure  6E, quantified in Figure 6G). Immunostaining for Ki-67 demonstrated that depletion of CCL2-driven monocytes/macrophages did not change the proliferation rate of AECs compared with β1 rtTA mice (Supplemental Figure 3, D and E). These findings indicate that the increase in epithelial cell numbers in β1 rtTA mice was due to impaired AEC removal rather than increased AEC proliferation. Despite numerous macrophages and an overabundance of type 2 AECs, there was almost no colocalization of CD68 and pro-SP-C in CCR2 -/-;β1 rtTA mice ( Figure 6H, quantified in Figure 6I), suggesting minimal efferocytosis in these mice. To directly test whether macrophages from CCR2 -/-;β1 rtTA mice were defective in efferocytosis, we collected macrophages from bronchoalveolar lavage and exposed these cells to fluorescently labeled primary type 2 AECs from β1 rtTA mice ( Figure 6J). While macrophages from β1 rtTA and β1 f/f lungs briskly engulfed β 1 -deficient AECs, macrophages from CCR2-deficient mice (both CCR2 -/-;β1 rtTA and CCR2 -/-;β1 f/f ) ingested far fewer labeled AECs, demonstrating that CCR2-deficient macrophages were less efficient efferocytosis agents. These data strongly suggest that the more severe phenotype in the CCR2 -/-;β1 f/f mice is caused by their inability to remove deranged type 2 AECS and that the efferocytosis function of CCL2-recruited macrophages is to limit inflammation and mitigate lung damage in β1 rtTA mice.
CD11b + CD11cmonocytes/macrophages efferocytose type 2 AECs in β1 rtTA mice. We next examined the immune cell population in the whole lung by flow cytometry. β1 rtTA lungs contained increased CD45 + CD-11b + CD11cimmune cells, markers consistent with recently recruited monocyte-early macrophages (Figure 7A, gating strategy in Supplemental Figure 4; and refs. 43,44). We identified this as a mixed population, as cells expressed the monocyte marker Ly6C, the macrophage marker CD64, or both ( Figure 7B). Since the CD11b + CD11cimmune cells were differentially enriched in β1 rtTA mice, we collected this population by FACS, cytospinned the cells, and immunostained for pro-SP-C and CD68 to determine whether these cells contributed to the increased efferocytosis seen in β1 rtTA mice. We found that 68% ± 4% of monocytes/macrophages collected from β1 rtTA lungs contained pro-SP-C + material compared with 14% ± 2% of these cells from β1 f/f lungs (Figure 7, C and D). To functionally phenotype these cells in β1 rtTA mice, we collected media from cultured monocytes/macrophages and assayed for cytokine production by cytokine multiplex. The β1 rtTA monocyte-macrophage population secreted only scant amounts of inflammatory cytokines/chemokines, equivalent to expression levels by cells from β1 f/f mice (Supplemental Table 1). Taken together, these data demonstrate that CD11b + CD11cmonocytes/macrophages are critical effector cells for efferocytosis but do not directly contribute to the inflammatory state of β1 rtTA mice.
β1 Integrin regulates AEC inflammation. Our data thus far suggest that the β1 integrin-null cells provide an inflammatory stimulus resulting in monocyte-macrophage chemoattraction into the alveolus. These recruited cells function as efferocytotic agents but do not contribute to the inflammatory status of the lungs. Immunostaining for pro-SP-C (green) and β 1 integrin (red) demonstrates type 2 AECspecific deletion of β 1 integrin in 3-month-old β1 rtTA lungs. Arrows indicate the presence/absence of β 1 integrin expression. Scale bar: 5 μm. (B) Type 2 AEC-specific deletion is represented as percentage of pro-SP-C + cells that express β 1 integrin. 100-120 type 2 AECs counted/mouse; n = 3 β1 f/f , n = 4 β1 rtTA mice. (C) Representative Western blot for β 1 integrin on primary type 2 AEC lysate, normalized to GAPDH; representative of 3 separate experiments. *P < 0.05 by 2-tailed Student's t test.
Next, we tested whether β 1 -deficient AECs drive the inflammatory phenotype in lungs of β1 rtTA mice. Ten of 32 cytokines (31%), including mediators of macrophage chemotaxis and maturation, were significantly increased in the culture media of β1 rtTA AECs compared with that of β1 f/f AECs ( Figure 8A and Supplemental Table 2). To define the consequence of increased AEC inflammatory signaling in the whole lung, we performed multiplex analysis on tissue lysates ( Figure 8B and Supplemental Table 3). Multiple inflammatory mediators were increased in lungs of β1 rtTA mice compared with β1 f/f controls. Even further increases were seen in CCR2 -/-;β1 rtTA , where inflamed β 1 -deficient type 2 AECs remained unchecked by efferocytosis. Since many of the cytokines increased in β1 rtTA and CCR2 -/-;β1 rtTA lungs were recognizable gene products of NF-κB signaling (including KC, IL-6, MIP-2, and G-CSF), we . Immunofluorescence staining for phospho-p65 (S276), a well-recognized marker of NF-κB activation (45), revealed numerous phospho-p65 + pro-SP-C + type 2 AECs in lungs from β1 rtTA mice and CCR2 -/-;β1 rtTA mice. Other cell types, in addition to type 2 AECs, exhibited NF-κB activation in β1 rt-TA and CCR2 -/-;β1 rtTA lungs. These findings indicate that β 1 integrin deficiency results in a pervasive inflammatory environment in the distal lung with contributions from retained β 1 -deficient type 2 AECs. β 1 -Deficient AEC inflammatory mediators are produced as a consequence of ROS generation. Since the generation of ROS has been linked to NF-κB-dependent cytokine expression in epithelial cells and β 1 -containing integrins have been shown to modulate ROS signaling (46-50), we measured ROS production in cultured type 2 AECs. We found that β1 rtTA type 2 AECs produced more superoxide (O 2 -) and hydrogen peroxide (H 2 O 2 ) than β1 f/f cells (Figure 9, A and B); however, no differences in mitochondria-derived ROS were detected ( Figure 9C). Given the increase in O 2 generation, we investigated whether the NADPH oxidase (NOX) system was upregulated in cells from β1 rtTA mice. Of the 5 major NADPH isoforms in the lung epithelium, Duox1 expression was markedly increased in freshly isolated β 1 -integrin deficient primary type 2 AECs, but there was no significant difference in expression of the NOX isoforms Duox2, Nox1, Nox2, or Nox4 between β 1 integrin-deficient and control cells ( Figure 9D).
To define whether ROS production stimulated NF-κB-dependent cytokine expression, we measured levels of CCL2, a known downstream cytokine product of NF-κB activation (51)(52)(53). We treated type 2 AECs isolated from β1 rtTA and β1 f/f mice with a superoxide dismutase mimetic (TEMPOL) or a pan-NOX inhibitor (DPI) and measured CCL2 concentration in the media by ELISA. Both TEMPOL and DPI treatment decreased CCL2 secretion by β1 rtTA type 2 AECs (Figure 9, E and F). Although a specific Duox1 inhibitor is not available, we narrowed down the NOX subunits potentially regulated by β 1 integrin using the NOX1/4 inhibitor GKT137831 ( Figure 9G). In contrast to the pan-NOX inhibitor DPI, treatment with GKT137831 did not reduce CCL2 secretion from β1 rtTA type 2 AECs, implicating NOX2, Duox1, and/or Duox2 as the source of increased ROS in β1 rtTA mice. As Duox1 was the only NOX isoform with increased expression, these data suggest that β 1 integrin regulates ROS production through this isoform in AECs.
To test whether ROS-dependent CCL2 production by β1 rtTA type 2 AECs was in part responsible for increased macrophage infiltration in β1 rtTA mice, we performed chemotaxis assays using WT macrophages collected by bronchoalveolar lavage and conditioned media from cultured type 2 AECs from β1 f/f and β1 rtTA mice. Macrophage migration toward media from β1 rtTA type 2 AECs was greatly enhanced compared with media from control cells, and this increase was completely abrogated by treatment with DPI or neutralizing antibodies to CCL2 ( Figure 9H). These findings support the conclusion that β1 rtTA type 2 AECs have persistent ROS production that contributes to CCL2 secretion that induces macrophage migration into the airspaces of β1 rtTA mice.

Discussion
While numerous studies have defined the critical role of integrins in organ morphogenesis, few have examined their role in tissue homeostasis in adults. In the setting of development, phenotype severity is highly correlated with timing of integrin deletion after conception and is primarily ascribed to defects in cell adhesion and migration. In this study, we defined the role of β 1 integrin in the structurally stable, fully formed alveolus of the lung, where epithelial cells undergo slow turnover and are tightly bound to the basement membrane. We show that deleting β 1 integrin in AECs under these circumstances results in emphysema, a condition characterized by destruction/loss of gas exchange units and chronic inflammation. Surprisingly, there were no adhesion defects in the AECs in our model; however, these cells were highly inflamed, with excessive ROS production that caused increased NF-κB-dependent cytokine production. Thus, β 1 integrin in alveolar epithelial cells has an antiinflammatory role and is required for alveolar homeostasis in the lung.
Our studies provide direct evidence that mice with a targeted deletion of β 1 integrin in type 2 AECs develop aging-related, spontaneous emphysema as quantified by mean linear intercept. This method easily captures one component of the emphysematous phenotype, enlargement of airspaces. Although we did not perform stereological analysis to address alveolar number specifically, we took precautions in our studies to minimize bias in our 2D morphological measurements from sampling (25)(26)(27). Loss of β 1 integrin in AECs stimulates ROS production and NF-κB signaling, and subsequently released inflammatory mediators recruit and activate a mixed population of monocytes/macrophages that efferocytose the β 1 -deficient AECs. One possible mechanism for the development of emphysema is that macrophages mediate lung destruction via altered protease/antiprotease balance (54)(55)(56). These observations are consistent with studies demonstrating a role for excessive ROS and NF-κB activation as initiators of macrophage accumulation and subsequent alveolar injury, resulting in emphysema (57)(58)(59). In addition, epithelial apoptosis in combination with ineffective efferocytosis could contribute to the development of emphysema. Both epithelial and endothelial apoptosis can contribute to emphysema independent of inflammation (60)(61)(62). Consistent with these potential explanations for development of emphysema in our model, blocking efferocytosis has been shown to potentiate alveolar destruction in murine models of elastase-induced emphysema associated with increased MMP2 and -12 expression (63). It is unclear whether loss of efferocytosis with its antiinflammatory effects or the retention of inflamed β 1 -deficient AECs causes emphysema. However, our data indicating that there is no phenotypical difference in the efferocytosing monocytes/macrophages suggest that retained β 1 -deficient AECs are the primary driver of emphysema in β1 rtTA mice. Our data also confirmed and extended studies that indirectly implicated β 1 -containing integrins in the pathogenesis of emphysema. Mice with impaired fucosylation exhibit an emphysematous lung phenotype, and fucosylation is required for normal α 3 β 1 integrin-dependent migration and signaling, suggesting that the phenotype is due to impairedα 3 β 1 integrin function (64,65). Similarly, fibulin 5 -/mice have enlarged airspaces at birth that progressively dilate into adulthood (66). Fibulin 5 is a ligand for α v β 3 -, α v β 5 , and α 9 β 1 integrins, participates in outside-in integrin signaling, and is crucial for proper assembly of elastic fibers (66, 67). We show that the inflammatory phenotype of β 1 integrin-deficient AECs, as manifested by increased NF-κB signaling and cytokine production, is mediated at least in part by excessive ROS production. While this phenomenon is well documented in multiple other cell types (68)(69)(70), the mechanisms whereby integrins regulate ROS production are poorly understood. Our studies implicate β 1 integrin as a critical negative regulator of the NOX isoform Duox1 in AECs. Previous studies reported that β 1 integrin negatively regulates ROS production through NOX2 in chondrocytes and kidney mesangial cells (46,47,71,72). Thus, integrins play a critical role in regulating ROS production in multiple cell types; however, the mechanisms appear to be cell type specific.
One of the most interesting observations in our study was that genetic depletion of CCR2, which blocks CCL2-mediated recruitment of monocyte-derived macrophages, exacerbates alveolar remodeling in adult β 1 -deficient mice. This contrasts with our previous observation that chemical depletion of macrophages using intranasal instillation of clodronate during lung development rescues alveolarization defects (22). These findings expose differential functions of macrophage subtypes and their potentially paradoxical roles in the adult versus developing lung. In development, fetal lung macrophages are essential for normal lung morphogenesis. They actively clear mesenchymal cells through phagocytosis during sacculation, and their response to inflammatory stimuli regulates airway branching through modulation of developmental signals (36)(37)(38)(39)(73)(74)(75). During homeostasis, macrophages are required for regulation of inflammatory signaling, host defense, and wound healing (76,77). The majority of efferocytosis activity following injury is accomplished by macrophages, but more recent data suggest that monocytes significantly contribute to efferocytosis and antigen presentation in the presence of apoptotic cells (78). Although monocytes and macrophages efferocytose dying cells during acute injury, their role in chronic inflammation is less well defined (78,79). In our model, deranged type 2 AECs are efferocytosed by the CD11b + CD11cmonocyte/macrophage population. This is likely a mix of newly recruited monocytes and monocytes transitioning into macrophages. In β1 rtTA mice, homeostatic compensation fails with loss of CCL2-driven monocyte-macrophage recruitment, resulting in an escalation of inflammation associated with diminished efferocytosis. Although determining why CCL2-recruited monocytes/macrophages are necessary for efficient efferocytosis of type 2 AECs will require further study, this finding could have direct implications for human lung diseases, including COPD, in which ineffective efferocytosis has been suggested to be a contributor to pathogenesis (80)(81)(82)(83)(84)(85)(86).
proliferation in the kidney, mammary, and submandibular glands (11,15,19,21,22), whereas increased epithelial proliferation has been reported when β 1 integrin was deleted in the intestine or skin (14,17). The mechanisms whereby β 1 integrin regulates cell number/density in fully formed organs is unknown; however, this could also be ROS mediated, since multiple investigations have shown that ROS can stimulate cell proliferation.
In conclusion, this study shows that loss of β 1 integrin in type 2 AECs promotes persistent lung inflammation and emphysematous remodeling, which is mitigated by efferocytosis of inflamed AECs by CD11b + CD11cmonocytes/macrophages. Thus, regulation of inflammation is the major function of β 1 integrin in alveolar homeostasis in the lung.

Methods
Mice. For timed deletion of β 1 integrin, we crossed transgenic mice with inducible Cre recombinase expression by the dox-inducible reverse tetracycline transactivator under control of the SP-C promoter (SP-C rtTA;Tet-O-Cre) with integrin β 1 fl/fl mice (95,96). Postdevelopmental type 2 AEC deletion was induced on P28 in these triple transgenic SP-C rtTA;Tet-O-Cre; β1 f/f mice (called β1 rtTA mice) using dox in drinking water (2 g/L × 4 weeks). Control littermate β1 f/f mice received identical dox treatment. To test the role of β 1 integrin in epithelial differentiation during alveolar homeostasis, we crossed β1 rtTA mice to the mTmG Cre recombinase reporter. To test the role of CCL2-recruited monocytes/macrophages in β1 integrin-regulated alveolar homeostasis, we crossed β1 rtTA and β1 f/f mice onto a homozygous null background for Ccr2, the CCL2 receptor. The resulting transgenic CCR2 -/-;SP-C rtTA;Tet-O-Cre;β1 f/f mice (termed CCR2 -/-;β1 rtTA mice) and control littermate CCR2 -/-;β1 f/f mice, received identical dox treatment on P28 to induce β 1 integrin deletion. Integrin β1 f/f mice were a gift from Elaine Fuchs (Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA). SP-C rtTA, Tet-O-Cre, Ccr2 homozygous null, and mTmG Cre recombinase reporter mice were purchased from the Jackson Laboratory. All mice were on a C57BL/6 background.
TEM. Lungs were harvested from 3-month-old β1 rtTA and β1 f/f mice, processed, postfixed with potassium ferrous cyanide, dehydrated with graded acetone, thick sectioned at 1 μm, thin sectioned at 80 nm in the region of interest, and imaged using a Philips FEI T-12 transmission electron microscope in the Vanderbilt Cell Imaging Shared Resource core. Bronchoalveolar lavage. Sterile saline lavages were performed with 1 mL PBS after sacrifice. Lavage fluid was centrifuged at 270 g at 4°C, and cells were resuspended and counted. The Pierce BCA Protein Assay kit (Thermo Fisher Scientific, 23225) was used to test for BALF protein per the manufacturer's instructions. For immunofluorescence analysis of immune cells collected by bronchoalveolar lavage, 40,000 cells were spun onto Shandon cytoslides (Thermo Fisher Scientific) at 240 g for 7 minutes, dried, and immunostained per the above protocol.
AEC isolation and collection of conditioned medium. Type 2 AECs were isolated from 3-month-old β1 rtTA and β1 f/f mice as previously described, yielding more than 90% type 2 AECs (22,97,98). Briefly, a single-cell suspension was generated with a 40-minute dispase digestion and 100-μm, 40-μm, and-20 μm serial filtration. The suspension was then incubated at 37°C for 2 hours in anti-CD45 (BD 553076) and anti-CD32 (BD 553142) antibody-coated plates for negative selection. The medium containing epithelial cells was collected and spun down, and AECs were plated in 5% bronchial epithelial cell growth medium (BEGM) on Matrigel-coated wells with or without the indicated treatment. Treatment reagents included TEMPOL (Sigma-Aldrich 176141) and DPI (Sigma-Aldrich D2926). Medium was collected at 24 hours for analysis.
ELISA and multiplex assay. ELISA for CCL2 and CX3CL1 on AEC conditioned media was performed in triplicate according to the manufacturer's instructions (R&D Systems, MJE00 and MCX310, respectively). Cytokine/chemokine Magnetic Bead 32-Multiplex Panel (Millipore MCYTMAG-70K-PX32) assay was performed on monocyte/macrophage conditioned media, AEC conditioned media, and whole lung tissue lysates in triplicate per the manufacturer's instructions. Tissue lysates were generated by sonicating the right upper lobe, centrifuging the tissue mixture, collecting the supernatant, and normalizing to protein. The Multiplex assay was read on the Luminex MAGPIX platform in the Vanderbilt Hormone and Analytical Services Core.
Efferocytosis assay. Macrophages collected by bronchoalveolar lavage (5 × 10 4 cells/well) were plated for 4 hours in serum-free media and exposed to fluorescently labeled primary type 2 AECs (Millipore 382065) from β1 rtTA mice (1 × 10 5 cell/well) for 1 hour. After incubation, nonadherent cells were removed by careful washing, and fluorescence was detected on a Molecular Devices SpectraMax M5 Plate Reader.
Macrophage migration assay. Conditioned medium from primary β1 rtTA and β1 f/f AECs was placed in the bottom chamber of a 5-μm Transwell insert (Corning 3422). WT macrophages (40,000 per insert) were obtained from pooled BALF and placed in the top chamber, incubated at 37°C for 4 hours. Unmigrated macrophages were removed from the top chamber, while migrated macrophages were fixed to the underside of the Transwell membrane and stained using the spHema 3 Manual Staining System