Airway level at which edema liquid enters the air space of isolated dog lungs

To identify lung units associated with liquid leakage into the air space in high-pressure pulmonary edema, we perfused air-inflated dog lung lobes with albumin solution to fill the loose peribronchovascular interstitium. Next, we perfused the lobes for 90 s with fluorescent albumin solution then froze the lobes in liquid nitrogen. This procedure confined the fluorescent perfusate to the liquid flux pathway between the circulation and the air space and eliminated the previously filled peribronchovascular cuffs as a source of the fluorescence that entered the air space. We divided each frozen lobe into three horizontal layers and prepared fluorescence-microscopic sections of each layer. In the most apical layers where alveolar flooding was minimal, 10.6 +/- 21.0% (SD) of alveolar ducts were either fluorescence filled or air filled and continuous with fluorescence-filled alveoli. In the same layers, 11.0 +/- 19.0% of respiratory bronchioles were similarly labeled. No terminal bronchioles in these layers were fluorescence labeled. This suggested that the fluorescent albumin entered the air space across the epithelium of respiratory bronchioles, alveolar ducts, or their associated alveoli. To simulate an alternative explanation, i.e., that fluorescence first entered central airways then flowed into peripheral air spaces, we prepared two additional lobes that we first partially inflated with fluorescent albumin then filled to capacity with air. This pushed the fluorescent solution along the airways into the lung periphery. In these lobes the ciliary lining of bronchi and terminal bronchioles was fluorescence coated. By comparison, cilia in fluorescence-perfused lobes were not coated. We conclude that alveolar flooding in hydrostatic pulmonary edema occurs across the epithelium of alveolar ducts, respiratory bronchioles, or their associated alveoli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fetal mouse lung in circumfusion system cultures.

Fetal mouse lungs were cultivated, using the dual-rotary circumfusion system for tissue culture, and their histotypic development was surveyed for 75 days by phase-contrast and electron microscopy. Alveoli, terminal bronchioles and alveolar macrophages were photographed periodically with still and time-lapse phase-contrast microscopy. Their histotypic appearance was confirmed by electron micrographs of the 1- and 2 1/2-month-old specimens. These revealed typical alveoli surrounded by a basal lamina and composed of types I and II pneumocytes containing various lamellar-body forms within the type II cells, the alveolar lumen, and the alveolar macrophages. There was a shift from almost all type II cells in the 1-month-old alveoli to the presence of frequent type I cells as constituents of the alveoli in the 2 1/2-month-old cultures. The terminal bronchioles were tubules consisting of ciliated cells with Clara cells interspersed between them. The ciliated cells contained as many as 30 cilia or basal bodies per section and numerous microvilli. They were attached to each other and to the Clara cells by junctional complexes and accessory desmosomes which were generally in the apical ends of the cells. The Clara cells typically had glycogen granules interspersed between lamellae of the endoplasmic reticulum, contained numerous well dispersed mitochondria, occasional lysosome-like granules and crystalloid bodies which appeared to be tubular. Some Clara cells presented a moderatley dense secretory granule in the center of the whorl of the endoplasmic reticulum.     

Fetal mouse lung in circumfusion system cultures

Summary Fetal mouse lungs were cultivated, using the dual-rotary circumfusion system for tissue culture, and their histotypic development was surveyed for 75 days by phase-contrast and electron microscopy. Alveoli, terminal bronchioles and alveolar macrophages were photographed periodically with still and time-lapse phase-contrast microscopy. Their histotypic appearance was confirmed by electron micrographs of the 1- and 2 1/2-month-old specimens. These revealed typical alveoli surrounded by a basal lamina and composed of types I and II pneumocytes containing various lamellar-body forms within the type II cells, the alveolar lumen, and the alveolar macrophages. There was a shift from almost all type II cells in the 1-month-old alveoli to the presence of frequent type I cells as constituents of the alveoli in the 2 1/2-month-old cultures. The terminal bronchioles were tubules consisting of ciliated cells with Clara cells interspersed between them. The ciliated cells contained as many as 30 cilia or basal bodies per section and numerous microvilli. They were attached to each other and to the Clara cells by junctional complexes and accessory desmosomes which were generally in the apical ends of the cells. The Clara cells typically had glycogen granules interspersed between lamellae of the endoplasmic reticulum, contained numerous well dispersed mitochondria, occasional lysosome-like granules and crystalloid bodies which appeared to be tubular. Some Clara cells presented a moderately dense secretory granule in the center of the whorl of the endoplasmic reticulum. This work supported by Grant HL19684 from the National Heart and Lung Institute, National Institutes of Health. Pregnant Strong A mice were kindly supplied by Dr. Henry Browning of the Department of Anatomy.     

Arguments against and alternatives for an extracellular surfactant layer in the alveoli of mammalian lung

It is generally believed that lung alveoli contain an extracellular aqueous layer of surfactant material, which is allegedly required to prevent alveolar collapse at small lung volume; the surfactant's major constituent is a fully saturated phospholipid, referred to as dipalmitoyl lecithin or DPL. I herein demonstrate that the surfactant hypothesis of alveolar stability is fundamentally wrong. Although DPL is synthesized inside type II epithelial cells and stored in the typical inclusion bodies therein and lowers surface tension to zero in the surface balance, there is no evidence to the effect that type II cells secrete the DPL surfactant into the aqueous intra-alveolar layer which is shown by electron microscopy in support of the surfactant theory. To the contrary, all the evidence indicates that, when seen, such an extracellular layer is an artifact. This is probably upon the damage glutaraldehyde inflicts onto alveolar structures during fixation of air-inflated lung tissue. Furthermore, several cogent arguments invalidate the belief that an extracellular layer of DPL and serum proteins is present in the alveoli of normal lung. In light of these arguments, a surface tension role of DPL in alveolar stability is excluded. Three hypotheses for an alternative role of DPL in respiration mechanics are proposed. They are: (a) alveolar clearance by viscolytic and surfactant action (bubble or foam formation) on the aqueous systems which are present in lung alveoli during edema and in prenatal life and which would otherwise be impervious to air; (b) homeostasis of blood palmitate in normal lung; (c) modulation of the elasticity of terminal lung tissue by the intact inclusion bodies and parts thereof inside type II cells in normal lung.     

TOPOGRAPHY OF DNA SYNTHESIS IN THE MAMMARY GLAND OF THE C3H MOUSE AND ITS CONTROL BY OVARIAN HORMONES: AN AUTORADIOGRAPHIC STUDY

Tritium-labelled thymidine was injected 45 min before sacrifice into virgin female C3H/HeJ mice 7–23 weeks of age, as well as into 10-week-old mice which had been ovariectomized and treated daily with 1 μg of oestradiol-17β and/or 1 mg of progesterone. Autoradiographs were made of squash preparations of the mammary glands, stained by Feulgen's method. The following results were obtained: (1) During normal development of the gland, cells synthesizing DNA are abundant in terminal buds and virtually absent in duct epithelium. Hence ductal growth takes place by the addition of cells produced in the terminal end structures. (2) At 5–6 months, when mammary growth has ceased, a considerable number of cells synthesizing DNA can still be found in alveoli, though not in duct epithelium. Hence the alveolar cells constitute a renewal population in the adult virgin. Because they maintain the potentiality to divide, duct cells are a G₀ population. (3) Ovariectomy results in arrest of DNA synthesis within 3–5 days. Both oestradiol and progesterone restore DNA synthesis in alveoli but only progesterone is able to induce DNA synthesis in duct epithelium, and the differentiation of terminal buds into alveoli. This finding provides an explanation for the resumed proliferation of duct cells in pregnancy. (4) The number of cells engaged in DNA synthesis varies considerably among identical structures within the same gland. This may be due either to synchrony of cell replication and/or to fluctuations of proliferative activity in the gland.     

Over-expression of an endogenous milk protein gene in transgenic mice is associated with impaired mammary alveolar development and a milchlos phenotype.

The whey acidic protein (WAP) gene is expressed in mammary epithelial cells at late pregnancy and throughout lactation. We have generated transgenic mice in which a mouse WAP transgene is expressed precociously in pregnancy. From 13 founder mice bearing WAP transgenes, two female founders and the daughters from a male founder failed to lactate and nurture their offspring. We named this phenotype milchlos. Mammary tissue from postpartum milchlos mice was underdeveloped, contained too few alveoli and resembled the glands of non-transgenic mid-pregnant mice. The hypothesis that alveolar development in milchlos mice was functionally arrested in a prelactational state is consistent with low levels of alpha-lactalbumin mRNA, and an unidentified keratin RNA in mammary tissue from postpartum mice. Defects in alveolar function in milchlos mice were detected at mid-pregnancy; in non-transgenic mice, WAP was secreted into the alveolar lumen but remained preferentially in the cytoplasm of the alveolar epithelial cells in the milchlos mice. Since deregulated WAP expression resulted in impaired mammary development, it is possible that WAP plays a regulatory role in the terminal differentiation and development of mammary alveolar cells.     

Effects of pulmonary surfactant and surfactant protein A on phagocytosis of fractionated alveolar macrophages: relationship to starvation.

Pulmonary surfactant isolated from bronchoalveolar lavage fluid of rat lung contained a high content of surfactant protein A (SP-A) in starved for 2 days compared to fed controls, but this phenomena returned to baseline following more than 4 days starvation. As determined by immunoperoxidase staining of lung sections using SP-A antibody, SP-A could be consistently observed in nonciliated bronchiolar (Clara) cells, alveolar type II cells and some alveolar macrophages (AM). Fc receptor-mediated phagocytosis of AM was enhanced by SP-A, which was dependent on the dosis and reached a maximum at 10 micrograms of SP-A/ml. Antibody to SP-A completely inhibited the enhanced response of phagocytosis. When exposed AM subpopulations, separated into four fractions (I, II, III and IV) by discontinuous Percoll gradient, to SP-A or pulmonary surfactant prepared from rats fed and starved for 2 days enhanced their phagocytic activity in high dense cells (III and IV), particularly to SP-A and pulmonary surfactant from rats starved for 2 days. Whereas little change in lower dense fractions (I and II) were seen in all exposures except for SP-A that enhanced the cells of fraction II. These results supported the concept that pulmonary surfactant and its apoprotein, SP-A, are a factor to regulate lung defense system including activation of AM that undergo different processes following starvation.     

Development of alveolar septa and formation of alveolar pores during the early postnatal period in the rat lung

In order to investigate the formation of alveolar pores, lungs of rats, after intratracheal perfusion of glutaraldehyde, are processed at postnatal days 1, 7, 14, 16 and 21 for light and transmission electron microscopy and at days 7 and 16 for scanning electron microscopy. The initial low secondary crests of day 1 rapidly elongate to pleats subdividing the primary saccules. The ledges of some pleats partly grow toward each other as ring like diaphragms, leaving openings whose boundary is composed of alveolar epithelium separated by a basal lamina from a connective tissue sheath with capillaries. At day 7, in scanning electron microscopy the lumina of some rudimentary alveoli communicate by apertures of different sizes, as a result of the outgrowth of curved alveolar pleats which narrow to a ring-like aperture. The interalveolar openings observed in scanning electron microscopy resemble those investigated by light and transmission electron microscopy. The number of interalveolar pores increases from day 7 on; they become more and more frequent at days 14, 16 and 21, respectively. It appears that alveolar multiplication in newborn rats proceeds not only by segmentation of terminal respiratory units but also by compoundment of septa. The difference between genuine pores and transsections of folds in transmission electron microscopy will be given closer attention in this study. Also, the incidence and location of type II pneumocytes during rapid enlargement of the alveolar surface area is discussed.     

The subgenus Persicargas (Ixodoidea: Argasidae: Argas). 23. Fine structure of the salivary glands of unfed A. (P.) Arboreus Kaiser, Hoogstraal, and Kohls.

Salivary glands of the unfed adult Argas (Persicargas) arboreus (family Argasidae) contain 2 types of alveoli, one nongranular and one granule-secreting. The fine structure of the nongranular alveolus is similar to that of the family Ixodidae. In the granule-secreting alveolus, the presence of 3 types of secretory cells, each with morphologically distinct granular inclusions, confirms histological and histochemical observations on argasid salivary glands. Epithelial cells with numerous membranous infoldings, mitochondria, microtubules, and a complex canalicular system probably concerned with fluid regulation and secretion are located between granule-secreting cells and form caps over their basal regions. The luminal border of both secretory and epithelial cells is microvillate. The alveolar lumen leads into the chitinous alveolar duct which lacks the complex valvular structure of ixodid alveoli. Axons containing neurosecretory material occur in both nongranular and granule-secreting alveoli and probably control salivary secretion.     

Morphologic aspects of alveolar microcirculation.

Direct observations of the flow direction and connections between arteries and veins in the mammal lung are difficult. When we divide the lung into smaller units like acini or segments we can observe a central supply of the unit with arterial blood that has venous drainage at different points of the periphery. Consideration of the situation prevailing at birth strongly suggests a preferential flow direction through paths located in primary septa at the bottom of alveoli. Capillaries of the secondary septa placed between alveoli open to the same duct represent collaterals of the mainstream flow filled only if pressure conditions permit. Another significant feature is the presence of pleated alveolar septa. While capillaries inside the interalveolar wall mostly appear flat or collapsed, the capillaries of the pleated alveolar corners are always wide open. Often they show openings into a small venule placed inside the pleated area, which strongly suggests that the pleated areas contain the venous side of the capillaries.     

Estrogen regulates pulmonary alveolar formation, loss, and regeneration in mice

Lung tissue elastic recoil and the dimension and number of pulmonary gas-exchange units (alveoli) are major determinants of gas-exchange function. Loss of gas-exchange function accelerates after menopause in the healthy aged and is progressively lost in individuals with chronic obstructive pulmonary disease (COPD). The latter, a disease of midlife and later, though more common in men than in women, is a disease to which women smokers and never smokers may be more susceptible than men; it is characterized by diminished lung tissue elastic recoil and presently irremediable alveolar loss. Ovariectomy in sexually immature rats diminishes the formation of alveoli, and estrogen prevents the diminution. In the present work, we found that estrogen receptor-alpha and estrogen receptor-beta, the only recognized mammalian estrogen receptors, are required for the formation of a full complement of alveoli in female mice. However, only the absence of estrogen receptor-beta diminishes lung elastic tissue recoil. Furthermore, ovariectomy in adult mice results, within 3 wk, in loss of alveoli and of alveolar surface area without a change of lung volume. Estrogen replacement, after alveolar loss, induces alveolar regeneration, reversing the architectural effects of ovariectomy. These studies 1) reveal estrogen receptors regulate alveolar size and number in a nonredundant manner, 2) show estrogen is required for maintenance of already formed alveoli and induces alveolar regeneration after their loss in adult ovariectomized mice, and 3) offer the possibility estrogen can slow alveolar loss and induce alveolar regeneration in women with COPD.     

Identification of Cell Types in the Developing Goat Mammary Gland

The goat was chosen as the model system for investigating mammary gland development in the ruminant. Histological and immunocytochemical staining of goat mammary tissue at key stages of development was performed to characterize the histogenesis of the ruminant mammary gland. The mammary gland of the virgin adult goat consisted of a ductal system terminating in lobules of ductules. Lobuloalveolar development of ductules occurred during pregnancy and lactation which was followed by the regression of secretory alveoli at involution. The ductal system was separated from the surrounding stroma by a basement membrane which was defined by antisera raised against laminin and Type IV collagen. Vimentin, smooth-muscle actin and myosin monoclonal antisera as well as antisera to cytokeratin 18 and multiple cytokeratins stained a layer of myoepithelial cells which surround the ductal epithelium. Staining of luminal epithelial cells by monoclonal antibodies to cytokeratins was dependent on their location along the ductal system, from intense staining in ducts to variable staining in ductules. The staining of epithelial cells by monoclonals to cytokeratins also varied according to the developmental status of the goat, being maximal in virgin and involuting glands, lowest at lactation and intermediate during gestation. In addition, cuboidal cells, situated perpendicular to myoepithelial cells and adjacent to alveolar cells in secretory alveoli, were also stained by cytokeratin monoclonal antibodies and antisera to the receptor protein, erbB-2, in similar fashion to luminal epithelial cells. These results demonstrate that caprine mammary epithelial cell differentiation along the alveolar pathway is associated with the loss of certain types of cytokeratins and that undifferentiated and secretory alveolar epithelial cells are present within lactating goat mammary alveoli.     

Use of peanut lectin and rat mammary stem cell lines to identify a cellular differentiation pathway for the alveolar cell in the rat mammary gland.

The presence of the carbohydrate receptor for PNL has been used to identify the previously described morphological types of epithelial cell produced as the stem cell line rat mammary 25 (Rama 25) differentiates to casein secretory alveolar-like cells in vitro. Thus when cultures of the epithelial stem cell line Rama 25 are treated with neuraminidase, fluorescently-conjugated PNL fails to stain cuboidal cells, stains weakly grey cells, and stains strongly the surface of dark cells. When superconfluent cultures of Rama 25 are treated with dimethyl sulfoxide or retinoic acid and prolactin, estradiol, hydrocortisone, and insulin to induce differentiation to alveolar cells, PNL stains strongly the untreated surfaces of droplet cells and casein-secreting vacuolated cells. PNL-staining of the derivative cell lines with truncated cellular pathways, and quantitative binding of [125I]-labeled PNL to the cultured cells are consistent with this cellular staining pattern. The presence of the carbohydrate receptor for peanut lectin (PNL) has also been used to identify specific epithelial cell types in different mammary structures of the developing rat mammary gland, as they differentiate to casein secretory alveolar cells in vivo. Thus when different structures of the developing rat mammary gland are treated with neuraminidase, peroxidase-conjugated PNL fails to stain histochemically the majority of epithelial cells in ducts, stains the cytoplasm of the majority of epithelial cells in terminal end-buds (TEBs), and stains strongly the luminal surfaces of the majority of epithelial cells in alveolar buds (ABs). PNL also stains the untreated luminal surfaces of alveolar cells, whether or not the cells can be stained with a monoclonal antibody to rat beta-casein. Stimulation of mammary differentiation by an analogue of ethyl retinoate or by perphenazine causes cells in end-buds to bind PNL without the necessity for their desialylation similar to that seen in casein secretory alveoli of lactating rats. In conclusion the different interconverting cell types of Rama 25 which form a pathway to casein-secretory cells in vitro are thus equated with recognisable epithelial cell types in vivo. These results suggest that casein-secretory cells in vivo are generated by similar successive interconversions between the major epithelial cell types present in the different mammary structures in the order: ducts, TEBs, ABs, alveoli, and secretory alveoli.     

Electron microscopical findings with special reference to cancer in rats caused by inhalation of nickel oxide

A special exposure system was used for the inhalation of nickel oxide (NiO) aerosol by Wistar male rats. The median aerodynamic diameter and the geometric standard deviation were 1.2 μm and 2.2, respectively. A histopathological study of the rats was performed immediately, and at intervals of 12 and 20 mo after a 1-mo expsoure to NiO. Electron microscopy showed that localization of NiO particles was restricted to the lungs and that each particle had been engulfed by the alveolar macrophages. Type II pneumocytes and nonciliated bronchiolar epithelial cells (Clara cells), as well as numerous tubular myelin (surfactant) in the alveoli were prominent. In rats dissected after 12 mo, clusters of NiO particles were still present within the terminal bronchioli, alveolar walls, and lysosomes of the alveolar macrophages. Pools of tubular myelin were observed in the peribron-chial lymphatics. The Clara cells, which project into the lumen of bronchioli, showed active secretion and were filled with smooth en-doplasmic reticulum (SER) in the apical cytoplasm. In the experimental group sacrificed after 20 mo, one rat had papillary adenocarcinoma and two rats showed adenomatosis in the peripheral portion of the lung, but none in the upper respiratory tract.     

Temporal expression of alpha-smooth muscle actin and drebrin in septal interstitial cells during alveolar maturation.

In rat lung, the definitive alveoli are established during development by the outgrowth of secondary septa from the primary septa present in newborn; however, the mechanism of alveolar formation has not yet been fully clarified. In this study, we characterize the septal interstitial cells in developing alveoli. During the perinatal period, alpha-SMA-containing slender cells were found in the primitive alveolar septa. Alpha-SMA-containing cells were detected at the tips of the septa until postnatal day 21, when the alveolar formation was almost completed, but disappeared in adult. Immunoelectron microscopy demonstrated that alpha-SMA is localized mainly in the cellular protrusions, which are connected with the elastic fibers around the interstitial cells. Developmentally regulated brain protein (drebrin) is also located in the cell extensions containing alpha-SMA in immature alveolar interstitial cells. In adult lung, alpha-SMA-positive cells are located only at the alveolar ducts but are not found in the secondary septa. Desmin is expressed only in alpha-SMA-containing cells at the alveolar ducts but not in those at the tip of alveolar septa. These results suggest that a part of the septal interstitial cells are temporarily alpha-SMA- and drebrin-positive during maturation. Alpha-SMA- and drebrin-containing septal interstitial cells (termed septal myofibroblast-like cells) may play an important role in alveolar formation.     

Calorie-related rapid onset of alveolar loss, regeneration, and changes in mouse lung gene expression

Calorie restriction, followed by ad libitum refeeding, results, respectively, in loss and regeneration of pulmonary alveoli. We now show 35% of alveoli are lost within 72 h of onset of calorie restriction ((2/3) decreased daily chow intake), and an additional 12% of alveoli are lost over a subsequent 12 days of calorie restriction. Tissue necrosis was not seen. Within 72 h of refeeding, after 15 days of calorie restriction, the number of alveoli returns to precalorie restriction values. Microarray lung gene profiling, in conjunction with Western and RNase protection assay, demonstrate an increase of granzyme and caspase gene expression 2-3 h after onset of calorie restriction. By 12 h, granzyme and caspase expression is no longer increased, but tumor necrosis factor death receptor expression is elevated. At 336 h, Fas death receptor expression is increased. Because granzymes are found only in cytotoxic lymphocytes (CTLs) and natural killer (NK) cells, we suggest calorie restriction activates these cells, initiating a series of molecular events that results in alveolar destruction. The evidence of involvement of CTLs and NK cells and the absence of necrosis are similar to alveolar destruction in chronic obstructive pulmonary disease.     

Reactive oxygen species initiate luminal but not basal cell death in cultured human mammary alveolar structures: a potential regulator of involution

Post-lactational involution of the mammary gland is initiated within days of weaning. Clearing of cells occurs by apoptosis of the milk-secreting luminal cells in the alveoli and through stromal tissue remodeling to return the gland almost completely to its pre-pregnant state. The pathways that specifically target involution of the luminal cells in the alveoli but not the basal and ductal cells are poorly understood. In this study we show in cultured human mammary alveolar structures that the involution process is initiated by fresh media withdrawal, and is characterized by cellular oxidative stress, expression of activated macrophage marker CD68 and finally complete clearing of the luminal but not basal epithelial layer. This process can be simulated by ectopic addition of reactive oxygen species (ROS) in cultures without media withdrawal. Cells isolated from post-involution alveoli were enriched for the CD49f⁺ mammary stem cell (MaSC) phenotype and were able to reproduce a complete alveolar structure in subcultures without any significant loss in viability. We propose that the ROS produced by accumulated milk breakdown post-weaning may be the mechanism underlying the selective involution of secretory alveolar luminal cells, and that our culture model represents an useful means to investigate this and other mechanisms further.     

Influenza virus-induced immune complexes suppress alveolar macrophage phagocytosis

Immune complexes in the lungs are capable of inducing adverse responses. Herein we have detailed the formation of immune complexes in the lungs of influenza virus-infected mice and examined their effect on alveolar macrophage defenses. On days 3, 7, 10, 15, and 30 after aerosol infection with influenza A/PR8/34 virus, the acellular pulmonary lavage fluid was tested for viral antigen, specific viral antibody, and immune complexes by immunoassays. Whereas peak viral antigen (day 3) diminished to undetectable levels by day 10, specific viral antibody remained at a low concentration until day 10, after which it rapidly increased. Immune complex concentrations increased through day 7, peaked at day 10, and gradually returned to the control level by day 30. These data demonstrate that immune complexes of detectable size are induced by influenza virus infection during the interface between antigen excess and antibody excess conditions. Since alveolar macrophages are the pivotal phagocytic defense cells in the lung, the ability of normal alveolar macrophages to ingest opsonized erythrocytes was quantitated in the presence of immune complexes from lavage fluid. Immune complexes from day 10 virus-infected lungs caused a dose-dependent suppression of antibody-mediated phagocytosis to 30% of control values. In contrast, although these immune complexes also markedly decreased the phagocytosis of antibody-coated yeast cells, they did not significantly impair the antibody-independent ingestion of unopsonized yeast cells by macrophages. the suppressive effects of immune complexes on alveolar macrophages may, in part, explain the phagocytic dysfunction that occurs 7 to 10 days after influenza virus pneumonia.     

Pulmonary surfactant protein A augments the phagocytosis of Streptococcus pneumoniae by alveolar macrophages through a casein kinase 2-dependent increase of cell surface localization of scavenger receptor A

Pulmonary surfactant proteins A (SP-A) and D (SP-D), members of the collectin family, play important roles in the innate immune system of the lung. Here, we show that SP-A but not SP-D augmented phagocytosis of Streptococcus pneumoniae by alveolar macrophages, independent of its binding to the bacteria. Analysis of the SP-A/SP-D chimeras, in which progressively longer carboxyl-terminal regions of SP-A were replaced with the corresponding SP-D regions, has revealed that the SP-D region Gly(346)-Phe(355) can be substituted for the SP-A region Leu(219)-Phe(228) without altering the SP-A activity of enhancing the phagocytosis and that the SP-A region Cys(204)-Cys(218) is required for the SP-A-mediated phagocytosis. Acetylated low density lipoprotein significantly reduced the SP-A-stimulated uptake of the bacteria. SP-A failed to enhance the phagocytosis of S. pneumoniae by alveolar macrophages derived from scavenger receptor A (SR-A)-deficient mice, demonstrating that SP-A augments SRA-mediated phagocytosis. Preincubation of macrophages with SP-A at 37 degrees C but not at 4 degrees C stimulated the phagocytosis. The SP-A-mediated enhanced phagocytosis was not inhibited by the presence of cycloheximide. SP-A increased cell surface localization of SR-A that was inhibitable by apigenin, a casein kinase 2 (CK2) inhibitor. SP-A-treated macrophages exhibited significantly greater binding of acetylated low density lipoprotein than nontreated cells. The SP-A-stimulated phagocytosis was also abolished by apigenin. In addition, SP-A stimulated CK2 activity. These results demonstrate that SP-A enhances the phagocytosis of S. pneumoniae by alveolar macrophages through a CK2-dependent increase of cell surface SR-A localization. This study reveals a novel mechanism of bacterial clearance by alveolar macrophages.