GM-CSF mediates alveolar macrophage proliferation and type II cell hypertrophy in SP-D gene-targeted mice

Mice deficient in surfactant protein (SP) D develop increased surfactant pool sizes and dramatic changes in alveolar macrophages and type II cells. To test the hypothesis that granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates alveolar macrophage proliferation and activation and the type II cell hypertrophy seen in SP-D null mice, we bred SP-D and GM-CSF gene-targeted mice to obtain littermate double null, single null, and wild-type mice. Bronchoalveolar lavage levels of phospholipid, protein, SP-D, SP-A, and GM-CSF were measured from 1 to 4 mo. There was an approximately additive accumulation of phospholipid, total protein, and SP-A at each time point. Microscopy showed normal macrophage number and morphology in GM-CSF null mice, numerous giant foamy macrophages and hypertrophic type II cells in SP-D null mice, and large but not foamy macrophages and mostly normal type II cells in double null mice. These results suggest that the mechanisms underlying the alveolar surfactant accumulation in the SP-D-deficient and GM-CSF-deficient mice are different and that GM-CSF mediates some of the macrophage and type II cell changes seen in SP-D null mice.     

Human insulin-like growth factor-IA expression in transgenic mice promotes adenomatous hyperplasia but not pulmonary fibrosis

Insulin-like growth factor-I (IGF-I) has been implicated in postnatal alveolar development, pulmonary fibrosis, and non-small cell lung cancer. To further investigate the role of IGF-I, we created a line of transgenic mice in which alveolar type II epithelial cells express human IGF-IA under the control of the surfactant protein C promoter. We determined the effect of pulmonary overexpression of human IGF-IA on 1) pulmonary inflammation and fibrosis in response to intratracheal instillation of bleomycin, 2) premalignant pulmonary adenomatous hyperplasia, and 3) adenoma formation. Transgenic expression of human IGF-IA had no effect on baseline gross lung pathology, cellularity of bronchoalveolar lavage, or total lung collagen content. In addition, there were no significant differences between transgenic mice and nontransgenic littermate controls in the development of pulmonary inflammation or pulmonary fibrosis in response to intratracheal bleomycin instillation. However, pulmonary expression of human IGF-IA in older mice (>12 mo) significantly increased the incidence of premalignant adenomatous hyperplastic lesions compared with littermate controls without affecting adenoma formation. These findings suggest that increased expression of human IGF-IA in alveolar air spaces does not affect the development of pulmonary fibrosis but promotes premalignant changes in the alveolar epithelium.     

Regulation of found in inflammatory zone 1 expression in bleomycin-induced lung fibrosis: role of IL-4/IL-13 and mediation via STAT-6

Found in inflammatory zone (FIZZ)1, also known as resistin-like molecule alpha, belongs to a novel class of cysteine-rich secreted protein family, named FIZZ/resistin-like molecule, with unique tissue expression patterns. FIZZ1 is induced in alveolar type II epithelial cells (AECs) in bleomycin (BLM)-induced lung fibrosis, and found to induce myofibroblast differentiation in vitro. The objective of this study was to elucidate the regulation of AEC FIZZ1 expression in pulmonary fibrosis. AECs were isolated from rat lungs and the effects of a number of cytokines on FIZZ1 expression were evaluated by RT-PCR. Of all cytokines examined, only IL-4 and IL-13 were effective in stimulating FIZZ1 expression in AECs. Stimulation by IL-4/IL-13 was accompanied by increases in phosphorylated STAT6 and JAK1. FIZZ1 expression was also stimulated by transfection with a STAT6 expression plasmid, but was inhibited by antisense oligonucleotides directed against STAT6. In vivo studies showed that compared with wild-type controls, both IL-4- and IL-13-deficient mice showed reduced BLM-induced lung FIZZ1 expression and fibrosis, which were essentially abolished in IL-4 and IL-13 doubly deficient mice. Furthermore, STAT6-deficient mice showed marked reduction in BLM-induced lung FIZZ1 expression. Thus, IL-4 and IL-13 are potent inducers of AEC FIZZ1 expression via STAT6 and play key roles in BLM-induced lung FIZZ1 expression and fibrosis. This represents a potential mechanism by which IL-4/IL-13 could play a role in the pathogenesis of lung fibrosis.     

Overexpression of tumor necrosis factor-alpha produces an increase in lung volumes and pulmonary hypertension

Tumor necrosis factor (TNF)-alpha is a key proinflammatory cytokine that is thought to be important in the development of pulmonary fibrosis, whereas its role in pulmonary emphysema has not been as thoroughly documented. In the present study, TNF-alpha was overexpressed in alveolar type II cells under the control of the human surfactant protein C promoter. In this report, we further characterized the pulmonary abnormalities and provided a physiological assessment of these mice. Histopathology of the lungs revealed chronic inflammation, severe alveolar air space enlargement and septal destruction, and bronchiolitis. However, pulmonary fibrosis was very limited and only seen in the subpleural, peribronchiolar, and perivascular regions. Physiological assessment showed an increase in lung volumes and a decrease in elastic recoil characteristic of emphysema; there was no evidence of restrictive lung disease characteristic of pulmonary fibrosis. In addition, the mice raised in ambient conditions in Denver developed pulmonary hypertension. Gelatinase activity was increased in the lavage fluid from these lungs. These results suggest that in these mice TNF-alpha contributed to the development of pulmonary emphysema through chronic lung inflammation and activation of the elastolytic enzymes but by itself was unable to produce significant pulmonary fibrosis.     

Impaired pulmonary defense against Pseudomonas aeruginosa in VEGF gene inactivated mouse lung

Repeated bacterial and viral infections are known to contribute to worsening lung function in several respiratory diseases, including asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). Previous studies have reported alveolar wall cell apoptosis and parenchymal damage in adult pulmonary VEGF gene ablated mice. We hypothesized that VEGF expressed by type II cells is also necessary to provide an effective host defense against bacteria in part by maintaining surfactant homeostasis. Therefore, Pseudomonas aeruginosa (PAO1) levels were evaluated in mice following lung‐targeted VEGF gene inactivation, and alterations in VEGF‐dependent type II cell function were evaluated by measuring surfactant homeostasis in mouse lungs and isolated type II cells. In VEGF‐deficient lungs increased PAO1 levels and pro‐inflammatory cytokines, TNFα and IL‐6, were detected 24 h after bacterial instillation compared to control lungs. In vivo lung‐targeted VEGF gene deletion (57% decrease in total pulmonary VEGF) did not alter alveolar surfactant or tissue disaturated phosphatidylcholine (DSPC) levels. However, sphingomyelin content, choline phosphate cytidylyltransferase (CCT) mRNA, and SP‐D expression were decreased. In isolated type II cells an 80% reduction of VEGF protein resulted in decreases in total phospholipids (PL), DSPC, DSPC synthesis, surfactant associated proteins (SP)‐B and ‐D, and the lipid transporters, ABCA1 and Rab3D. TPA‐induced DSPC secretion and apoptosis were elevated in VEGF‐deficient type II cells. These results suggest a potential protective role for type II cell‐expressed VEGF against bacterial initiated infection. J. Cell. Physiol. 228: 371–379, 2013. © 2012 Wiley Periodicals, Inc.     

Anorexigen-induced pulmonary hypertension and the serotonin (5-HT) hypothesis: lessons for the future in pathogenesis

Pulmonary surfactant is a surface active material composed of both lipids and proteins that is produced by alveolar type II pneumocytes. Abnormalities of surfactant in the immature lung or in the acutely inflamed mature lung are well described. However, in a variety of subacute diseases of the mature lung, abnormalities of lung surfactant may also be of importance. These diseases include chronic obstructive pulmonary disease, asthma, cystic fibrosis, interstitial lung disease, pneumonia, and alveolar proteinosis. Understanding of the mechanisms that disturb the lung surfactant system may lead to novel rational therapies for these diseases.     

GM-CSF mediates alveolar epithelial type II cell changes, but not emphysema-like pathology, in SP-D-deficient mice

Surfactant protein D (SP-D) is a member of the collectin subfamily of C-type lectins, pattern recognition proteins participating in the innate immune response. Gene-targeted mice deficient in SP-D develop abnormalities in surfactant homeostasis, hyperplasia of alveolar epithelial type II cells, and emphysema-like pathology. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is required for terminal differentiation and subsequent activation of alveolar macrophages, including the expression of matrix metalloproteinases and reactive oxygen species, factors thought to contribute to lung remodeling. Type II cells also express the GM-CSF receptor. Thus we hypothesized GM-CSF might mediate some or all of the cellular and structural abnormalities in the lungs of SP-D-deficient mice. To test this, SP-D (D-G+) and GM-CSF (D+G-) single knockout mice as well as double knockout mice deficient for both SP-D and GM-CSF (D-G-) were analyzed by design-based stereology. Compared with wild type, D-G+ as well as D+G- mice showed decreased alveolar numbers, increased alveolar sizes, and decreased alveolar epithelial surface areas. These emphysema-like changes were present to a greater extent in D-G- mice. D-G+ mice developed type II cell hyperplasia and hypertrophy with increased intracellular surfactant pools, whereas D+G- mice had smaller type II cells with decreased intracellular surfactant pools. In contrast to the emphysematous changes, the type II cell alterations were mostly corrected in D-G- mice. These results indicate that GM-CSF-dependent macrophage activity is not necessary for emphysema development in SP-D-deficient mice, but that type II cell metabolism and proliferation are, either directly or indirectly, regulated by GM-CSF in this model.     

Lung surfactant in subacute pulmonary disease

Pulmonary surfactant is a surface active material composed of both lipids and proteins that is produced by alveolar type II pneumocytes. Abnormalities of surfactant in the immature lung or in the acutely inflamed mature lung are well described. However, in a variety of subacute diseases of the mature lung, abnormalities of lung surfactant may also be of importance. These diseases include chronic obstructive pulmonary disease, asthma, cystic fibrosis, interstitial lung disease, pneumonia, and alveolar proteinosis. Understanding of the mechanisms that disturb the lung surfactant system may lead to novel rational therapies for these diseases.     

Lung infection with gamma-herpesvirus induces progressive pulmonary fibrosis in Th2-biased mice

Idiopathic pulmonary fibrosis (IPF) is a progressive, fibrotic lung disease of unknown etiology. A viral pathogenesis in IPF has been suggested since >95% of IPF patients have evidence of chronic pulmonary infection with one or more herpesviruses. To determine whether pulmonary infection with herpesvirus can cause lung fibrosis, we infected mice with the murine gamma-herpesvirus 68 (MHV68). Because IPF patients have a T helper type 2 (Th2) pulmonary phenotype, we used IFN-gammaR-/-, a strain of mice biased to develop Th2 responses. Chronic MHV68 infection of IFN-gammaR-/- mice resulted in progressive deposition of interstitial collagen as shown by light and electron microscopy. A significant decrease in tidal volume paralleled the collagen deposition. Five features typically seen in IPF, increased transforming growth factor-beta expression, myofibroblast transformation, production of Th2 cytokines, hyperplasia of type II cells, and increased expression of matrix metalloproteinase-7, were also present in chronically infected IFN-gammaR-/- mice. There also was altered synthesis of surfactant proteins, which is seen in some patients with familial IPF. MHV68 viral protein was found in type II alveolar epithelial cells, especially in lung areas with extensive alveolar remodeling. In summary, chronic herpesvirus pulmonary infection in IFN-gammaR-/- mice causes progressive pulmonary fibrosis and many of the pathological features seen in IPF.     

Dose-dependent lung remodeling in transgenic mice expressing transforming growth factor-alpha

Transgenic mice overexpressing human transforming growth factor-alpha (TGF-alpha) develop emphysema and fibrosis during postnatal alveologenesis. To assess dose-related pulmonary alterations, four distinct transgenic lines expressing different amounts of TGF-alpha in the distal lung under control of the surfactant protein C (SP-C) promoter were characterized. Mean lung homogenate TGF-alpha levels ranged from 388 +/- 40 pg/ml in the lowest expressing line to 1,247 +/- 33 pg/ml in the highest expressing line. Histological assessment demonstrated progressive alveolar airspace size changes that were more severe in the higher expressing TGF-alpha lines. Pleural and parenchymal fibrosis were only detected in the highest expressing line (line 28), and increasing terminal airspace area was associated with increasing TGF-alpha expression. Hysteresis on pressure-volume curves was significantly reduced in line 28 mice compared with other lines of mice. There were no differences in bronchoalveolar lavage fluid cell count or differential that would indicate any evidence of lung inflammation among all transgenic lines. Proliferating cells were increased in line 28 without alterations of numbers of type II cells. We conclude that TGF-alpha lung remodeling in transgenic mice is dose dependent and is independent of pulmonary inflammation.     

Role of bone marrow-derived mesenchymal stem cells in the prevention of hyperoxia-induced lung injury in newborn mice

BPD (bronchopulmonary dysplasia) is predominantly characterized by persistent abnormalities in lung structure and arrested lung development, but therapy can be palliative. While promising, the use of BMSC (bone marrow-derived mesenchymal stem cell) in the treatment of lung diseases remains controversial. We have assessed the therapeutic effects of BMSC in vitro and in vivo. In vitro co-culturing with injured lung tissue increased the migration-potential of BMSC; and SP-C (surfactant protein-C), a specific marker of AEC2 (type II alveolar epithelial cells), was expressed. Following intraperitoneal injection of BMSC into experimental BPD mice on post-natal day 7, it was found that BMSC can home to the injured lung, express SP-C, improve pulmonary architecture, attenuate pulmonary fibrosis and increase the survival rate of BPD mice. This work supports the notion that BMSC are of therapeutic benefit through the production of soluble factors at bioactive levels that regulate the pathogenesis of inflammation and fibrosis following hyperoxia.     

Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis

Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ), provides antifibrotic effects without systemic toxicity in a mouse model of lung fibrosis. Oprozomib was first tested on the human alveolar epithelial cancer cell line A549 and in primary mouse alveolar epithelial type II cells regarding its cytotoxic effects on alveolar epithelial cells and compared to the FDA approved proteasome inhibitor bortezomib (BZ). OZ was less toxic than BZ and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, OZ showed significant anti-fibrotic effects, i.e. reduction of collagen I and α smooth muscle actin expression, in the absence of cytotoxicity. When applied locally into the lungs of healthy mice via instillation, OZ was well tolerated and effectively reduced proteasome activity in the lungs. In bleomycin challenged mice, however, locally applied OZ resulted in accelerated weight loss and increased mortality of treated mice. Further, OZ failed to reduce fibrosis in these mice. While upon systemic application OZ was well tolerated in healthy mice, it rather augmented instead of attenuated fibrotic remodelling of the lung in bleomycin challenged mice. To conclude, low toxicity and antifibrotic effects of OZ in pulmonary fibroblasts could not be confirmed for pulmonary fibrosis of bleomycin-treated mice. In light of these data, the use of proteasome inhibitors as therapeutic agents for the treatment of fibrotic lung diseases should thus be considered with caution.     

A profibrotic function of IL-12p40 in experimental pulmonary fibrosis

The p40 subunit of IL-12 (IL-12p40), but not the heterodimeric form IL-12p70, is secreted during the development of silica-induced lung fibrosis in C57BL/6 mice. To delineate the contribution of IL-12p40 to the lung inflammatory and fibrotic processes, we compared the pulmonary responses with silica particles of IL-12p35-deficient mice (IL-12p35(-/-), able to produce IL-12p40) and IL-12p40-deficient mice (IL-12p40(-/-)). IL-12p35(-/-) and IL-12p40(-/-) animals developed strikingly contrasting responses to silica in comparison with wild-type C57BL/6 mice. Although the IL-12p40(-/-) mice exhibited limited inflammatory and fibrotic reactions, the IL-12p35(-/-) mice presented a robust and well-developed pulmonary inflammation and fibrosis. Furthermore, the silica-induced increase in lung IL-12p40 content was significantly higher in IL-12p35(-/-) mice than in wild-type controls, and was associated with extensive lung fibrosis and pulmonary macrophage infiltration. The contrasting responses observed between these two IL-12 subunit-deficient murine strains were not accompanied by a strict type 1 or type 2 polarization as estimated by the measurements of lung IFN-gamma/IgG2a and IL-4/IgG1 content. In vitro proliferation, type I collagen expression, as well as myofibroblast differentiation of purified pulmonary fibroblasts were not affected by treatment with exogenous rIL-12p40. In vivo, supplementation with rIL-12p40 restored the impaired pulmonary fibrotic response and macrophage accumulation in silica-treated IL-12p40(-/-) mice, and also promoted fibrosis and macrophage influx in wild-type mice. Together, our data suggest that IL-12p40 plays an important role in silica-induced pulmonary inflammation and fibrosis, possibly by exacerbating macrophage recruitment.     

Bleomycin-induced lung fibrosis in IL-4-overexpressing and knockout mice

The role of IL-4 in the development of lung fibrosis is as yet unclear. Bleomycin (Bleo) or saline (Sal) was injected intratracheally into three groups of C57BL/6J mice: transgenic animals that overexpressed IL-4 (IL-4 TG, n = 14), mice with a targeted knockout mutation of the IL-4 gene (IL-4 KO, n = 11), and wild-type (WT, n = 13) mice. At 14 days, lung fibrosis was evaluated by hydroxyproline measurement and by quantitative image analysis of fibrosis fraction and alveolar wall area fraction. Bronchoalveolar lavage cell counts in all Bleo-treated groups demonstrated an increased percentage of lymphocytes with a corresponding decrease in the percentage of macrophages. Comparing Bleo- to Sal-treated controls within each group of mice showed increases in all lung fibrosis parameters in IL-4 KO and WT, but not in any of the parameters in IL-4 TG mice. The severity of Bleo-induced fibrotic response was decreased in overexpressed IL-4 TG compared with IL-4 KO mice. These data negate a critical profibrotic role for IL-4 in Bleo-induced lung fibrosis.     

NMDA receptor activation induces damage of alveolar type II cells and lung fibrogenesis through ferroptosis

Ferroptosis, a newly discovered type of regulated cell death, has been implicated in numerous human diseases. Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal interstitial lung disease with poor prognosis and limited treatment options. Emerging evidence has linked ferroptosis and glutamate-determined cell fate which is considered a new light on the etiology of pulmonary fibrosis. Here, we observed that N-methyl d-aspartate receptor (NMDAR) activation promoted cell damage and iron deposition in MLE-12 cells in a dose-, time-, and receptor-dependent manner. This mediated substantial Ca²⁺ influx, upregulated the expression levels of nNOS and IRP1, and affected intracellular iron homeostasis by regulating the expression of iron transport-related proteins (i.e., TFR1, DMT1, and FPN). Excessive iron load promoted the continuous accumulation of total intracellular and mitochondrial reactive oxygen species, which ultimately led to ferroptosis. NMDAR inhibition reduced lung injury and pulmonary fibrosis in bleomycin-induced mice. Bleomycin stimulation upregulated the expression of NMDAR1, nNOS, and IRP1 in mouse lung tissues, which ultimately led to iron deposition via regulation of the expression of various iron metabolism-related genes. NMDAR activation initiated the pulmonary fibrosis process by inducing iron deposition in lung tissues and ferroptosis of alveolar type II cells. Our data suggest that NMDAR activation regulates the expression of iron metabolism-related genes by promoting calcium influx, increasing nNOS and IRP1 expression, and increasing iron deposition by affecting cellular iron homeostasis, ultimately leading to mitochondrial damage, mitochondrial dysfunction, and ferroptosis. NMDAR activation-induced ferroptosis of alveolar type II cells might be a key event to the initiation of pulmonary fibrosis.     

Effect of inhaled crystalline silica in a rat model: time course of pulmonary reactions

Numerous investigations have been conducted to elucidate mechanisms involved in the initiation and progression of silicosis. However, most of these studies involved bolus exposure of rats to silica, i.e. intratracheal instillation or a short duration inhalation exposure to a high dose of silica. Therefore, the question of pulmonary overload has been an issue in these studies. The objective of the current investigation was to monitor the time course of pulmonary reactions of rats exposed by inhalation to a non-overload level of crystalline silica. To accomplish this, rats were exposed to 15 mg/m3 silica, 6 h/day, 5 days/week for up to 116 days of exposure. At various times (5-116 days exposure), animals were sacrificed and silica lung burden, lung damage, inflammation, NF-KB activation, reactive oxygen species and nitric oxide production, cytokine production, alveolar type II epithelial cell activity, and fibrosis were monitored. Activation of NF-KB/DNA binding in BAL cells was evident after 5 days of silica inhalation and increased linearly with continued exposure. Parameters of pulmonary damage, inflammation and alveolar type II epithelial cell activity rapidly increased to a significantly elevated but stable new level through the first 41 days of exposure and increased at a steep rate thereafter. Pulmonary fibrosis was measurable only after this explosive rise in lung damage and inflammation, as was the steep increase in TNF-alpha and IL-1 production from BAL cells and the dramatic rise in lavageable alveolar macrophages. Indicators of oxidant stress and pulmonary production of nitric oxide exhibited a time course which was similar to that for lung damage and inflammation with the steep rise correlating with initiation of pulmonary fibrosis. Staining for iNOS and nitrotyrosine was localized in granulomatous regions of the lung and bronchial associated lymphoid tissue. Therefore, these data demonstrate that the generation of oxidants and nitric oxide, in particular, is temporally and anatomically associated with the development of lung damage, inflammation, granulomas and fibrosis. This suggests an important role for nitric oxide in the initiation of silicosis.     

Inhibition of NF-kappaB activation reduces the tissue effects of transgenic IL-13

IL-13 is a major Th2 cytokine that is capable of inducing inflammation, excessive mucus production, airway hyperresponsiveness, alveolar remodeling, and fibrosis in the murine lung. Although IL-13 through its binding to IL-4Ralpha/IL-13Ralpha1 uses the canonical STAT6-signaling pathway to mediate these tissue responses, recent studies have demonstrated that other signaling pathways may also be involved. Previous studies from our laboratory demonstrated that IL-13 mediates its tissue effects by inducing a wide variety of downstream genes many of which are known to be regulated by NF-kappaB. As a result, we hypothesized that NF-kappaB activation plays a critical role in the pathogenesis of IL-13-induced tissue alterations. To test this hypothesis, we compared the effects of transgenic IL-13 in mice with normal and diminished levels of NF-kappaB activity. Three pharmacologic approaches were used to inhibit NF-kappaB including 1) PS1145, a small molecule inhibitor of IkappaBalpha kinase (IKK2), 2) antennapedia-linked NF-kappaB essential modulator-binding domain (NBD) peptide (wild-type NBD), and 3) an adenoviral construct expressing a dominant-negative version of IKK2. We also crossed IL-13-transgenic mice with mice with null mutations of p50 to generate mice that overproduced IL-13 in the presence and absence of this NF-kappaB component. These studies demonstrate that all these interventions reduced IL-13-induced tissue inflammation, fibrosis and alveolar remodeling. In addition, we show that both PS1145 and wild-type NBD inhibit lung inflammatory and structural cell apoptosis. PS1145 inhibits caspase activation and up-regulates inhibitor of apoptosis protein cellular-inhibitor of apoptosis protein 1 (c-IAP-1). Therefore, NF-kappaB is an attractive target for immunotherapy of IL-13-mediated diseases.     

Comparison of effects of epidermal and insulin-like growth factors,gastrin releasing peptide and retinoic acid on fetal lung cell growth and maturation in vitro

The role in cell multiplication and maturation of several factors present in the late fetal lung was explored on isolated fetal rat pulmonary fibroblasts and alveolar epithelial type II cells cultivated in serum-free medium. The low degree of reciprocal contamination of each cell population was assessed by immunocytochemistry. Epidermal Growth Factor (EGF) stimulated thymidine incorporation and DNA accumulation in both cell types. In type II cells, it increased labeled-choline incorporation into surfactant phosphatidylcholine (PC), consistently with previous data obtained with lung explant cultures, but not into non-surfactant PC. Insulin-like growth factor (IGF)-I slightly stimulated DNA accumulation in fibroblasts although it did not significantly stimulate thymidine incorporation, contrary to IGF-II which presented a dose-dependent stimulating activity of thymidine incorporation. Neither IGF-I nor IGF-II stimulated type II cell growth. IGFs thus appear to primarily control the growth of lung mesenchyme. In type II cells, they stimulated the most non-surfactant PC biosynthesis. Gastrin releasing peptide (GRP) which was recently reported to promote fetal lung growth in vivo and to stimulate surfactant biosynthesis in lung organ culture revealed as a growth factor for type II cells only, at concentrations below 10 ⁻⁹ M. At concentration 10 ⁻⁸ M, although it did not affect DNA synthesis, GRP tended to increase surfactant and non-surfactant-PC biosynthesis. Retinoic acid inhibited thymidine incorporation into type II cells on a dose-dependent manner but nevertheless enhanced surfactant-PC biosynthesis to a similar extent as EGF. It is suggested that retinoic acid may represent a differentiation or maturation factor for the alveolar epithelium.