Immunological dysregulation of lung cells in response to vitamin E deficiency

Vitamin E supplementation exhibits anti-inflammatory properties. In the lung, the beneficial effects of vitamin E supplementation on inflammation and infections are well documented, but potential consequences of alimentary vitamin E deficiency to the immunological status of lung cells are not known. It is unclear if temporary vitamin E deficiency exhibits deleterious consequences or can be compensated for by other cellular antioxidants. To address this question, the alimentary vitamin E supply to rats was modified. We then investigated the effects on major histocompatibility molecule (MHC) class II, cell adhesion molecules, interleukin (IL)10, tumor necrosis factor (TNF)alpha in various lung cells. The constitutive expression of MHC class II, intercellular adhesion molecule (ICAM)-1, L-selectin, alpha5-integrin, and CD 166, was demonstrated by flow cytometry on type II pneumocytes, alveolar macrophages, and on co-isolated lymphocytes. Vitamin E depletion increased ICAM-1 and CD166 on type II cells and macrophages, whereas the expression of L-selectin increased only on macrophages. Furthermore, the vitamin E depletion increased the cellular content and secretion of IL10 in type II cells, but decreased the content and secretion of TNFalpha. Vitamin E depletion decreased the cellular vitamin E content, but did not change the activity of antioxidant enzymes (catalase, superoxide dismutase) and the glutathion (GSH)/oxidized glutathion (GSSG) ratio in alveolar type II cells. The shift of protein kinase C (PKC) from the cytosol to membranes indicates that a PKC-dependent signaling pathway may be involved in the change of the immunological status of type II cells. All these effects were reversed by vitamin E repletion. In summary, these results are clearly compatible with the view that a temporary vitamin E deficiency induces a reversible immunological dysregulation in alveolar type II cells and lung macrophages. This deficiency might predispose the lung to develop acute or chronic inflammation.     

Vitamin E deficiency reduces surfactant lipid biosynthesis in alveolar type II cells

Reactive oxygen species play an important role in development of lung injury. Neonates exhibit a high risk of developing acute and/or chronic lung disorder, often associated with surfactant deficiency, and in parallel they show low vitamin E concentration. We investigated whether the vitamin E status of adult rats affects the content of phospholipids (PL) in bronchoalveolar lavage and alveolar type II cells. Phosphatidylcholine (PtdCho) is the dominant and functional most important PL in lung surfactant. Therefore, we determined its formation via de novo synthesis and reacylation of lyso-PtdCho in type II cells. Vitamin E depletion caused a decrease of PL content in bronchoalveolar lavage and type II cells and decreased glycerol-3-phosphate O-acyltransferase (G3P-AT) activity, de novo synthesis of PtdCho, and reacylation of lyso-PtdCho in type II cells. Preincubation of type II cell homogenates with dithiothreitol restored the activity of G3P-AT and de novo synthesis but inhibited reacylation. Reacylation was strongly reduced by chelerythrine-mediated inhibition of protein kinase C. We conclude that antioxidant and PKC-modulating properties of vitamin E regulate de novo synthesis of PtdCho and reacylation of lyso-PtdCho in alveolar type II cells. Vitamin E depletion reduced the two pathways of PL synthesis and caused a decrease of PL content in alveolar surfactant of rats.     

Nitric oxide decreases surfactant protein gene expression in primary cultures of type II pneumocytes

Inhaled nitric oxide (NO) is a selective pulmonary vasodilator effective in treating persistent pulmonary hypertension in newborns and in infants following congenital heart disease surgery. Recently, multiple in vivo and in vitro studies have shown a negative effect of NO on surfactant activity as well as surfactant protein gene expression. Although the relationship between NO and surfactant has been studied previously, the data has been hard to interpret due to the model systems used. The objective of the current study was to characterize the effect of NO on surfactant protein gene expression in primary rat type II pneumocytes cultured on a substratum that promoted the maintenance of type II cell phenotype. Exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP), decreased surfactant protein (SP)-A, (SP)-B, and (SP)-C mRNA levels in type II pneumocytes in a time- and dose-dependent manner. The effect was mediated in part by an increase in endothelin-1 secretion and a decrease in the intracellular messenger, phosphorylated ERK1/2 mitogen-activated protein kinases (MAPK). Exposing type II pneumocytes to endothelin-1 receptor antagonists PD-156707 or bosentan before exposure to SNAP partially prevented the decrease in surfactant protein gene expression. The results showed that NO mediated the decrease in surfactant protein gene expression at least in part through an increase in endothelin-1 secretion and a decrease in phosphorylated ERK1/2 MAPKs.     

High-avidity monoclonal antibodies against the human scavenger class B type I receptor efficiently block hepatitis C virus infection in the presence of high-density lipoprotein

The human scavenger class B type 1 receptor (SR-B1/Cla1) was identified as a putative receptor for hepatitis C virus (HCV) because it binds to soluble recombinant HCV envelope glycoprotein E2 (sE2). High-density lipoprotein (HDL), a natural SR-B1 ligand, was shown to increase the in vitro infectivity of retroviral pseudoparticles bearing HCV envelope glycoproteins and of cell culture-derived HCV (HCVcc), suggesting that SR-B1 promotes viral entry in an HDL-dependent manner. To determine whether SR-B1 participates directly in HCV infection or facilitates HCV entry through lipoprotein uptake, we generated a panel of monoclonal antibodies (MAbs) against native human SR-B1. Two of them, 3D5 and C167, bound to conformation-dependent SR-B1 determinants and inhibited the interaction of sE2 with SR-B1. These antibodies efficiently blocked HCVcc infection of Huh-7.5 hepatoma cells in a dose-dependent manner. To examine the role of HDL in SR-B1-mediated HCVcc infection, we set up conditions for HCVcc production and infection in serum-free medium. HCVcc efficiently infected Huh-7.5 cells in the absence of serum lipoproteins, and addition of HDL led to a twofold increase in infectivity. However, the HDL-induced enhancement of infection had no impact on the neutralization potency of MAb C167, despite its ability to inhibit both HDL binding to cells and SR-B1-mediated lipid transfer. Of note, MAb C167 also potently blocked Huh-7.5 infection by an HCV strain recovered from HCVcc-infected chimpanzees. These results demonstrate that SR-B1 is essential for infection with HCV produced in vitro and in vivo and suggest the possible use of anti-SR-B1 antibodies as therapeutic agents.     

Acylation of lysine residues in human plasma high density lipoprotein increases stability and plasma clearance in vivo

Although human plasma high density lipoproteins (HDL) concentrations negatively correlate with atherosclerotic cardiovascular disease, underlying mechanisms are unknown. Thus, there is continued interest in HDL structure and functionality. Numerous plasma factors disrupt HDL structure while inducing the release of lipid free apolipoprotein (apo) AI. Given that HDL is an unstable particle residing in a kinetic trap, we tested whether HDL could be stabilized by acylation with acetyl and hexanoyl anhydrides, giving AcHDL and HexHDL respectively. Lysine analysis with fluorescamine showed that AcHDL and HexHDL respectively contained 11 acetyl and 19 hexanoyl groups. Tests with biological and physicochemical perturbants showed that HexHDL was more stable than HDL to perturbant-induced lipid free apo AI formation. Like the reaction of streptococcal serum opacity factor against HDL, the interaction of HDL with its receptor, scavenger receptor class B member 1 (SR-B1), removes CE from HDL. Thus, we tested and validated the hypothesis that selective uptake of HexHDL-[³H]CE by Chinese Hamster Ovary cells expressing SR-B1 is less than that of HDL-[³H]CE; thus, selective SR-B1 uptake of HDL-CE depends on HDL instability. However, in mice, plasma clearance, hepatic uptake and sterol secretion into bile were faster from HexHDL-[³H]CE than from HDL-[³H]CE. Collectively, our data show that acylation increases HDL stability and that the reaction of plasma factors with HDL and SR-B1-mediated uptake are reduced by increased HDL stability. In vivo data suggest that HexHDL promotes charge-dependent reverse cholesterol transport, by a mechanism that increases hepatic sterol uptake via non SR-B1 receptors, thereby increasing bile acid output.     

Vitamin E deficiency sensitizes alveolar type II cells for apoptosis

Pre-term neonates and neonates in general exhibit physiological vitamin E deficiency and are at increased risk for the development of acute lung diseases. Apoptosis is a major cause of acute lung damage in alveolar type II cells. In this paper, we evaluated the hypothesis that vitamin E deficiency predisposes alveolar type II cells to apoptosis. Therefore, we measured markers of apoptosis in alveolar type II cells isolated from control rats, vitamin E deficient rats and deficient rats that were re-fed a vitamin E-enriched diet. Bax and cytosolic cytochrome c increased, and the mitochondrial transmembrane potential and Hsp25 expression was reduced in vitamin E deficiency. Furthermore, increased DNA-fragmentation and numbers of early and late apoptotic cells were seen, but caspases 3 and 8 activities and expression of Fas, Bcl-2, Bcl-x and p53 remained unchanged. Vitamin E depletion did not change the GSH/GSSG ratio and the activities of antioxidant enzymes. Thus, vitamin E deficiency may induce a reversible pro-apoptotic response in lung cells and sensitise them for additional insult. In agreement with this hypothesis, we demonstrate that in vivo hyperoxia alone does not induce apoptosis in type II cells of control rats but reversibly increases DNA-fragmentation and numbers of early apoptotic type II cells in vitamin E-depleted cells.     

High-throughput screening of bacteriorhodopsin mutants in whole cell pastes

Pre-term neonates and neonates in general exhibit physiological vitamin E deficiency and are at increased risk for the development of acute lung diseases. Apoptosis is a major cause of acute lung damage in alveolar type II cells. In this paper, we evaluated the hypothesis that vitamin E deficiency predisposes alveolar type II cells to apoptosis. Therefore, we measured markers of apoptosis in alveolar type II cells isolated from control rats, vitamin E deficient rats and deficient rats that were re-fed a vitamin E-enriched diet. Bax and cytosolic cytochrome c increased, and the mitochondrial transmembrane potential and Hsp25 expression was reduced in vitamin E deficiency. Furthermore, increased DNA-fragmentation and numbers of early and late apoptotic cells were seen, but caspases 3 and 8 activities and expression of Fas, Bcl-2, Bcl-x and p53 remained unchanged. Vitamin E depletion did not change the GSH/GSSG ratio and the activities of antioxidant enzymes. Thus, vitamin E deficiency may induce a reversible pro-apoptotic response in lung cells and sensitise them for additional insult. In agreement with this hypothesis, we demonstrate that in vivo hyperoxia alone does not induce apoptosis in type II cells of control rats but reversibly increases DNA-fragmentation and numbers of early apoptotic type II cells in vitamin E-depleted cells.     

B类1型清道夫受体表达调控与信号转导通路

B类1型清道夫受体(scavenger receptor class B type 1,SR-B1)是一种与清道夫受体CD36具有高度同源性的膜糖蛋白,其表达相对广泛且有着众多生物学作用.体内外多种因素可从转录或转录后水平对SR-B1表达进行调控： PPARα/γ激动剂、部分LXR激动剂、LH/HCG、雌激素等能上调SR-B1的表达;维生素E、INFα、脂多糖、IGF-1、胆酸、PXR激动剂及高糖水平等能下调SR-B1的表达;而血管紧张素Ⅱ则可对SR-B1的表达进行双向调节,且它们具体的调节机制复杂.SR-B1作为一种具有多配体结合特性的膜受体,不同配体与其结合后可介导细胞内不同信号事件及生物学效应,如介导HDL激活细胞内PI3K/Akt及MAPK信号途径, 增加内皮型一氧化氮合酶的磷酸化、促进内皮细胞迁移与内皮重构.此外,非HDL类配体如LDL激活p38MAPK途径、凋亡细胞、血清淀粉样蛋白A等激活胞内MAPK途径均可由SR-B1介导.本文对近年来B类1型清道夫受体表达调控机制及信号转导通路的相关研究进行综述.     

Scavenger receptor class B,type 1 facilitates cellular fatty acid uptake

SR-B1 belongs to the class B scavenger receptor, or CD36 super family. SR-B1 and CD36 share an affinity for a wide array of ligands. Although they exhibit similar ligand binding specificity, SR-B1 and CD36 have some very specific lipid transport functions. Whereas SR-B1 primarily facilitates the selective delivery of cholesteryl esters (CEs) and cholesterol from HDL particles to the liver and non-placental steroidogenic tissues, as well as participating in cholesterol efflux from cells, CD36 primarily mediates the uptake of long-chain fatty acids in high fatty acid-requiring organs such as the heart, skeletal muscle and adipose tissue. However, CD36 also mediates cholesterol efflux and facilitates selective lipoprotein-CE delivery, although less efficiently than SR-B1. Interestingly, the ability or efficiency of SR-B1 to mediate fatty acid uptake has not been reported. In this paper, using overexpression and siRNA-mediated knockdown of SR-B1, we show that SR-B1 possesses the ability to facilitate fatty acid uptake. Moreover, this function is not blocked by BLT-1, a specific chemical inhibitor of HDL-CE uptake activity of SR-B1, nor by sulfo-N-succinimidyl oleate, which inhibits fatty acid uptake by CD36. Attenuated fatty acid uptake was also observed in primary adipocytes isolated from SR-B1 knockout mice. In conclusion, facilitation of fatty acid uptake is an additional function that is mediated by SR-B1.     

Dehydroepiandrosterone fatty acyl esters in high density lipoprotein: interaction with human vascular endothelial cells and vascular responses ex vivo

Dehydroepiandrosterone (DHEA) fatty acyl esters once incorporated in high density lipoprotein (HDL) induce a stronger vasodilatory response in rat mesenteric arteries ex vivo compared to native HDL. We studied the role of HDL receptor, scavenger receptor class B, type 1 (SR-B1), as well as estrogen and androgen receptors in the vasodilatory response of HDL-associated DHEA fatty acyl esters. Using cultured human vascular endothelial cells (HUVEC), we investigated the possible internalization and cellular response of HDL-associated DHEA esters. We prepared DHEA ester-enriched HDL by incubating human plasma in the presence of DHEA. After isolation and purification, HDL was added in cumulative doses to arterial rings precontracted with noradrenaline. Inhibition of the function of SR-B1 almost completely abolished maximal vasorelaxation by DHEA-enriched HDL while estrogen or androgen receptor blockage had no significant effect. When HUVECs were incubated in the presence of [³H]DHEA ester-enriched HDL, the amount of intracellular [³H]-radioactivity increased steadily during 24 h. Blocking of SR-B1 reduced this uptake by a mean of 30%. The proportion of unesterified [³H]DHEA, as analyzed by thin-layer chromatography, increased intracellularly and in the cell culture media after several hours of incubation of the cells in the presence of [³H]DHEA ester-enriched HDL. This indicated slow hydrolysis of DHEA fatty acyl esters and subsequent excretion of unesterified DHEA by the cells. In conclusion, DHEA-enriched HDL induced vasorelaxation via the SR-B1-facilitated pathway. However, this vasodilation is not likely to be attributed to rapid hydrolysis of HDL-associated DHEA esters by the vascular endothelium.     

Rat Leydig cells use apolipoprotein E depleted high density lipoprotein to regulate testosterone production

Rat HDL are known to increase testosterone production by cultured Leydig cells either following gonadotropin stimulation or cholesteryl ester depletion. However, rat HDL contain apolipoprotein E and have a high affinity for the members of the low density receptor family such as LDL receptor, LDL receptor related protein and VLDL receptor. In contrast with the adrenal cells, the contribution of apo A-I and apo E pathways in HDL cholesterol uptake has not been yet evidenced in rat Leydig cells. Recent data provided evidence that hCG stimulates scavenger receptor BI expression in testes. In order to investigate if testosterone production can be stimulated by apo E depleted HDL, we compared the level of testosterone stimulation by HDL with or without apo E first, in presence of saturating dose of hCG (1 IU/ml) and second, after depletion of cholesterol synthesis by pravastatin, an inhibitor of HMG-CoA reductase. In presence of hCG, HDL with or without apo E increased testosterone production respectively by 37 and 25%. Pravastatin at 100 g/ml inhibited the cholesterol synthesis and the testosterone production by 25% and decreased the cholesteryl content by 25%. The addition of HDL with or without apo E (50 g protein HDL/ml) completely overcame the depletion of cellular cholesteryl esters and the inhibition of testosterone production induced by pravastatin. In the presence of heparin, apo E depleted HDL overcame the testosterone production induced by pravastatin, indicating that uptake of HDL without apo E via a secretion of apo E by the cells themselves was not involved. Therefore, in absence of apo E, it is suggested that rat Leydig cells used HDL to regulate steroidogenesis via an apolipoprotein A-I pathway.     

Interaction of the human prostacyclin receptor with the PDZ adapter protein PDZK1: role in endothelial cell migration and angiogenesis

Prostacyclin is increasingly implicated in re-endothelialization and angiogenesis but through largely unknown mechanisms. Herein the high-density lipoprotein (HDL) scavenger receptor class B, type 1 (SR-B1) adapter protein PDZ domain-containing protein 1 (PDZK1) was identified as an interactant of the human prostacyclin receptor (hIP) involving a Class I PDZ ligand at its carboxyl terminus and PDZ domains 1, 3, and 4 of PDZK1. Although the interaction is constitutive, it may be dynamically regulated following cicaprost activation of the hIP through a mechanism involving cAMP-dependent protein kinase (PK)A-phosphorylation of PDZK1 at Ser-505. Although PDZK1 did not increase overall levels of the hIP, it increased its functional expression at the cell surface, enhancing ligand binding and cicaprost-induced cAMP generation. Consistent with its role in re-endothelialization and angiogenesis, cicaprost activation of the hIP increased endothelial cell migration and tube formation/in vitro angiogenesis, effects completely abrogated by the specific IP antagonist RO1138452. Furthermore, similar to HDL/SR-B1, small interfering RNA (siRNA)-targeted disruption of PDZK1 abolished cicaprost-mediated endothelial responses but did not affect VEGF responses. Considering the essential role played by prostacyclin throughout the cardiovascular system, identification of PDZK1 as a functional interactant of the hIP sheds significant mechanistic insights into the protective roles of these key players, and potentially HDL/SR-B1, within the vascular endothelium.     

Stimulation of pulmonary surfactant secretion by activating neutrophils in rat type II pneumocytes culture

The influence of activating neutrophils on the secretion of phosphatidylcholine (PC), the predominant component of pulmonary surfactant, was examined using primary culture of rat type II pneumocytes. Simultaneous addition of neutrophils and opsonized zymosan, but not neutrophils or opsonized zymosan alone, to type II pneumocytes caused a significant increase in PC secretion without affecting the release of lactate dehydrogenase, a marker of cytotoxicity. The increase in PC secretion was dependent on the number of activating neutrophils. In addition, pretreatment of culture with the combination of superoxide dismutase and catalase inhibited the increase in PC secretion. These findings indicate that activating neutrophils stimulate the secretion of pulmonary surfactant and that the stimulation is mediated by oxygen radicals.     

Vitamin E differentially regulates the expression of peroxiredoxin-1 and -6 in alveolar type II cells

Vitamin E is the primary lipophilic antioxidant in mammals. Lack of vitamin E may lead to an increase of cytotoxic phospholipid-peroxidation products (PL-Ox). However, we could previously show that alimentary vitamin E-depletion in rats did not change the concentrations of dienes, hydroperoxides, and platelet-activating factor-related oxidation products in alveolar type II cells (TII cells). We hypothesized that vitamin E deficiency increases the activity of enzymes involved in the degradation of PL-Ox. Degradation of PL-Ox may be catalyzed by phospholipase A2, PAF-acetylhydrolase, or peroxiredoxins (Prx's). Alimentary vitamin E deficiency in rats increased the expression of Prx-1 at the mRNA and protein levels and the formation of Prx-SO3, but it did not change the expression of Prx-6 or the activity of phospholipase A2 and PAF-acetylhydrolase in TII cells. H2O2-induced oxidative stress in isolated TII cells activated protein kinase Calpha (PKCalpha) and increased the expression of Prx-1 and Prx-6. Inhibition of PKCalpha in isolated TII cells by long-time incubation with PMA inhibited PKCalpha and Prx-1 but not Prx-6. We concluded that the expression of Prx-1 and -6 is selectively regulated in TII cells; PKCalpha regulates the expression of Prx-1 but not Prx-6. Prx-6 expression may be closely linked to lipid peroxidation.     

Presenilin-1 processing of ErbB4 in fetal type II cells is necessary for control of fetal lung maturation

Maturation of pulmonary fetal type II cells to initiate adequate surfactant production is crucial for postnatal respiratory function. Little is known about specific mechanisms of signal transduction controlling type II cell maturation. The ErbB4 receptor and its ligand neuregulin (NRG) are critical for lung development. ErbB4 is cleaved at the cell membrane by the γ-secretase enzyme complex whose active component is either presenilin-1 (PSEN-1) or presenilin-2. ErbB4 cleavage releases the 80 kDa intracellular domain (4ICD), which associates with chaperone proteins such as YAP (Yes-associated protein) and translocates to the nucleus to regulate gene expression. We hypothesized that PSEN-1 and YAP have a development-specific expression in fetal type II cells and are important for ErbB4 signaling in surfactant production. In primary fetal mouse E16, E17, and E18 type II cells, PSEN-1 and YAP expression increased at E17 and E18 over E16. Subcellular fractionation showed a strong cytosolic and a weaker membrane location of both PSEN-1 and YAP. This was enhanced by NRG stimulation. Co-immunoprecipitations showed ErbB4 associated separately with PSEN-1 and with YAP. Their association, phosphorylation, and co-localization were induced by NRG. Confocal immunofluorescence and nuclear fractionation confirmed these associations in a time-dependent manner after NRG stimulation. Primary ErbB4-deleted E17 type II cells were transfected with a mutant ErbB4 lacking the γ-secretase binding site. When compared to transfection with wild-type ErbB4, the stimulatory effect of NRG on surfactant protein mRNA expression was lost. We conclude that PSEN-1 and YAP have crucial roles in ErbB4 signal transduction during type II cell maturation.     

Caveolin-1 does not affect SR-BI-mediated cholesterol efflux or selective uptake of cholesteryl ester in two cell lines

Free cholesterol (FC) has been reported to efflux from cells through caveolae, which are 50-100 nm plasma membrane pits. The 22 kDa protein caveolin-1 is concentrated in caveolae and is required for their formation. The HDL scavenger receptor BI (SR-BI), which stimulates both FC efflux and selective uptake of HDL-derived cholesteryl ester (CE), has been reported to be concentrated in caveolae, suggesting that this localization facilitates flux of FC and CE across the membrane. However, we found that overexpression of caveolin-1 in Fischer rat thyroid (FRT) cells, which lack caveolin-1 and caveolae, or HEK 293 cells, which normally express very low levels of caveolin-1, did not affect FC efflux to HDL or liposomes. Transient expression of SR-B1 did not affect this result. Similarly, caveolin-1 expression did not affect selective uptake of CE from labeled HDL particles in FRT or HEK 293 cells transfected with SR-BI. We conclude that basal and SR-BI-stimulated FC efflux to HDL and liposomes and SR-BI-mediated selective uptake of HDL CE are not affected by caveolin-1 expression in HEK 293 or FRT cells.     

HSL-knockout mouse testis exhibits class B scavenger receptor upregulation and disrupted lipid raft microdomains

There is a tight relationship between fertility and changes in cholesterol metabolism during spermatogenesis. In the testis, class B scavenger receptors (SR-B) SR-BI, SR-BII, and LIMP II mediate the selective uptake of cholesterol esters from HDL, which are hydrolyzed to unesterified cholesterol by hormone-sensitive lipase (HSL). HSL is critical because HSL knockout (KO) male mice are sterile. The aim of the present work was to determine the effects of the lack of HSL in testis on the expression of SR-B, lipid raft composition, and related cell signaling pathways. HSL-KO mouse testis presented altered spermatogenesis associated with decreased sperm counts, sperm motility, and infertility. In wild-type (WT) testis, HSL is expressed in elongated spermatids; SR-BI, in Leydig cells and spermatids; SR-BII, in spermatocytes and spermatids but not in Leydig cells; and LIMP II, in Sertoli and Leydig cells. HSL knockout male mice have increased expression of class B scavenger receptors, disrupted caveolin-1 localization in lipid raft plasma membrane microdomains, and activated phospho-ERK, phospho-AKT, and phospho-SRC in the testis, suggesting that class B scavenger receptors are involved in cholesterol ester uptake for steroidogenesis and spermatogenesis in the testis.     

Depletion of alveolar glycogen corresponds with immunohistochemical development of CD208 antigen expression in perinatal lamb lung.

CD208 DC lysosomal-associated protein is a marker of activated human dendritic cells; however, recently it was described as a marker of adult type II pneumocytes in many species including humans and sheep. Our hypothesis was that CD208 is developmentally regulated in lung pneumocytes. Lamb lungs at varying stages of development were stained immunohistochemically for CD208 and with Nile red (a fluorescent stain for lamellar bodies of type II cells) along with pulmonary markers of maturation (glycogen stores and surfactant protein A [SP-A] expression) or proliferation (Ki-67). CD208 staining and Nile red were localized to rare pneumocytes in young fetal lambs (day 115), increasing in frequency and stain intensity with age. Periodic acid-Schiff staining of glycogen granules was most prominent in the young lambs (day 115) with reduced staining through advancing lung development. SP-A was detected in pulmonary epithelia and staining in alveoli increased through gestation with decreased staining at 2 weeks of age. Intranuclear Ki-67 staining decreased through late gestation but was increased in 2-week-old lambs. Ontogeny of CD208 staining and depletion of glycogen were correlated (p<0.0001) and consistent with the premise that CD208 is localized to developing lamellar bodies. The findings suggest that CD208 antigen expression may serve as a marker for pneumocyte maturation in the developing fetal lung.