Nitric oxide production by rat alveolar macrophages can be modulated in vitro by surfactant protein A.

Alveolar macrophage and type II cells are known to generate nitric oxide, which is a highly reactive molecule that plays a role in host defense against pathogens, as well as tissue damage associated with inflammation in the lung. Both types of cells are known to generate the nitric oxide by inducible nitric oxide synthase (iNOS). Surfactant-associated protein A (SP-A) from various sources (human alveolar proteinosis, rat and recombinant rat) was found to upregulate nitric oxide production by alveolar macrophages in a concentration- and time-dependent manner, whereas type II cells were unresponsive to SP-A. The increase in nitric oxide production was associated with elevation in the expression of iNOS. However, only 30-50% of the cells responded by expressing iNOS, as was observed by immunofluorescence staining. The stimulatory effect of SP-A was found to be 30-50% lower than the known nitric oxide agonists interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). However, addition of the cytokines interleukin-1 or granulocyte macrophage colony-stimulating factor elevated the levels of nitric oxide production to that of LPS and IFN-gamma. Special attention was given to exclude the possibility that contaminating LPS in the various SP-A species stimulated nitric oxide production by the macrophages. Our results indicate that SP-A is the agonist and not a contaminating LPS. The data presented in this report extend our knowledge regarding the nonsurfactant-related functions of SP-A.

Interaction of surfactant protein A with bacterial lipopolysaccharide may affect some biological functions.

Cultured alveolar type II cells and alveolar macrophages were found to secrete colony-stimulating factors (CSF) into the medium. Surfactant protein A (SP-A; 0.1-5 micrograms/ml) and bacterial lipopolysaccharide (LPS; 10-20 micrograms/ml) were found to upregulate the secretion of CSF (seven-fold) from these cells. However, a reversal of the stimulatory effect was observed when the two agents were added simultaneously to the cells. SP-A-enhanced phagocytosis of bacteria by alveolar macrophages was also inhibited by simultaneous addition of SP-A and LPS. Thus some biological activities attributed to either SP-A or LPS are inhibited in the simultaneous presence of the two agents. We therefore investigated the possibility of interaction and binding between SP-A and LPS molecules. Our biochemical data that include immunoblots and enzyme-linked immunosorbent assay support the notion that SP-A is capable of binding LPS, and this interaction is time and concentration dependent. The binding was partially inhibited (60%) by antibody to SP-A. The binding was calcium independent and was not affected by excess carbohydrates such as methyl alpha-D-mannopyranoside or heparin. Lipid A, the hydrophobic component of LPS, however, inhibited the SP-A-LPS interaction and also caused a partial reversal of the binding. Thus these results indicate that lipid A is associated with this binding. The biological implication of SP-A-LPS interaction, especially during inflammatory responses, is discussed.

Secretion of cytokines by rat alveolar epithelial cells: possible regulatory role for SP-A.

Cultured alveolar type II cells and pulmonary epithelial (PE) cells in long-term culture were found to secrete colony-stimulating factors (CSF) into the medium in similar fashion to alveolar macrophages. CSF activity was determined by using the in vitro assay for myeloid progenitor cells [colony-forming units in culture (CFU-C)]. Both lipopolisaccharide (LPS) and interleukin-1 alpha (IL-1 alpha) were found to upregulate the secretion 6.5- to 8-fold from alveolar type II cells and macrophages. However, no stimulatory effect of these factors was observed in PE cells that release CSF into the medium constitutively, possibly due to the conditions of long-term culture. The CSF activity was partially neutralized (70% inhibition) by antibodies against murine granulocyte/macrophage (GM)-CSF and IL-3, thus indicating the presence of both GM-CSF and IL-3-like factors in the CSF. However, the presence of other cytokines in the CSF is highly probable. Surfactant-associated protein A (SP-A), which is known to play a central role in surfactant homeostasis and function, was also found to upregulate secretion of CSF (at concentrations of 0.1-5 micrograms/ml) from alveolar type II cells and macrophages. Control cells such as rat peritoneal macrophages, alveolar fibroblasts, and 3T3/NIH cell line could not be elicited by SP-A to release CSF. The results are discussed in relation to the possible participation of the alveolar epithelial cells in various intercellular signaling networks. Our studies suggest that alveolar type II cells and SP-A may play an important regulatory role in the modulation of immune and inflammatory effector cells within the alveolar space.

Pulmonary epithelial cell proliferation in primary culture of alveolar type II cells.

A small subpopulation of pulmonary epithelial cells (PE) proliferates in low-density primary culture of alveolar type II cells and forms colonies of cells that could be passaged for several generations and that in some respects maintain a differentiated phenotype of the alveolar type II cells. At this time it is not known if these cells are some form of progenitor epithelial cells or type II cells that are not fully differentiated in vitro. The proliferation of the PE cells was dependent on serum, alveolar macrophage-conditioned medium, and insulin being included in the culture medium. Under these conditions, approximately 0.5-1.0% of the seeded cells that adhered to the culture dishes were capable of forming colonies. Efficiency of colony formation increased to 5-10% in subsequent passages. PE cells maintained a high level (> 40%) of saturated phosphatidylcholine (PC) as a percentage of total PC throughout the culture period (> 28 days). However, the saturated PC content was not constant throughout the long-term culture period and the subsequent passages (41.3% at 29 days and 37.3% in the 3rd passage). These cells also contained numerous lamellar bodies and were able to bind the Maclura pomifera lectin. PE cells also expressed cytokeratin No. 19, as well as alkaline phosphatase activity, both possible markers for differentiated type II cells. However, PE cell synthesized low levels of Pg (approximately 2%), were squamous, and tended to form multiple strata, unlike the cuboidal type II cells in vivo. The cells did not exhibit immunocytochemically demonstrable surfactant-associated protein A (SP-A). Additional factors and culture requirements may be necessary for complete maturation of cultured PE cells. This was demonstrated by culturing PE cells on EHS matrix. Aggregates of cells surrounding a central lumen were formed after a few hours in culture and were maintained for 20 days. The cells contained lamellar bodies and some intercellular junctions. PE cells can be regarded as a highly selected subpopulation of pulmonary epithelial cells that concomitantly maintain proliferation and aspects of differentiated alveolar type II cells in long-term culture.

Modulation of alkaline phosphatase activity in alveolar type II like cells.

Alveolar type II like cells (ALT II) represent a small subpopulation of alveolar type II cells, which is able to proliferate, can be passaged and possess many characteristics of differentiated adult type II cells. A correlation was found between the growth and development of ALT II cells in culture and their alkaline phosphatase activity. Unlike alveolar type II cells, which lose the activity in culture, ALT II cells regain the activity and maintain it for a long culture period. Quantitative histochemical analysis of the stained cells indicate that 80% of the cells at days 15-20 in culture are alkaline phosphatase positive. Inhibition studies indicate that alkaline phosphatase from ALT II cells and freshly isolated type II cells were similar. The inhibition of ALT II alkaline phosphatase by L-levamisole and its heat stability are similar to that of the bone enzyme and differ from the intestinal enzyme. Alkaline phosphatase expression is considered part of the differentiated phenotype of these cells. Therefore, the presence of this enzyme in ALT II cells adds support to the notion that these cells maintain many aspects of mature alveolar type II cells.

Alveolar type II-like cell colonies: effect of alveolar macrophages and macrophage-conditioned media.

Alveolar type II-like colonies were obtained after a low density plating (5 X 10(3)/60 mm tissue culture dish) of primary type II cells. These colonies were formed only when type II cells were either cocultured with alveolar macrophages or with conditioned media generated by alveolar macrophages. Cells in the colonies appeared homogeneous and kept their lamellar bodies over a period of 8 weeks and more, as observed by electron microscopy. These cells reacted immunocytochemically with antibodies directed against the 32-38 kDa protein fractions of rat surfactant.

Cytochemical approach to the development and behaviour of rat alveolar type II cells in culture.

The development and characteristics of rat alveolar type II cells were monitored by using various cytochemical techniques. Polarized light microscopy was found useful for observing live type II cells in culture. Cells progressively lose their birefringent granules starting from 48 h of cells in culture, indicating the disappearance of the phospholipids organized lamellae in the lamellar bodies. Similar results were obtained by using an immunocytochemical approach with antibodies raised against the apoprotein component of rat surfactant. A progressive decrease in immuno-staining corresponded to the disappearance of the lamellar bodies, and birefringence. Changes in lectins binding to the cultured type II cells were also observed. Freshly isolated and one day old cultured cells could bind Macula pomifera (MPA) but not Arachis hypogaea (PA) lectins. The reverse was found in 6-7 days old cultured cells which had the ability to bind PA but not MPA the advantage of using various cytochemical techniques for studying the development of type II cells in culture is being discussed.

The catabolism of lung surfactant by alveolar macrophages.

Surfactant was isolated from lung tissue of normal and chlorocyclizine-fed rats. Chlorocyclizine surfactant contained 2.5-3.4 times more phospholipids per mg protein than normal surfactant. Alveolar macrophages, incubated in vitro with normal and chlorocyclizine surfactants hydrolyzed the surfactant phospholipids and incorporated the fatty acids into cellular triacylglycerol. Employing [3H]palmitate-labeled surfactant, it was shown that cells incubated with chlorocyclizine surfactant incorporated 46.2-73.0 nmol of fatty acids per mg protein and were transformed into foam cells. Employing fluorescein or 125I-labeled surfactant, the uptake of surfactant protein by macrophages was shown. No significant differences between protein uptake from normal and chlorocyclizine surfactants were observed. These results suggest that the surfactant phospholipids and protein were catabolized independently.

The effect of chloroquine upon the developing lens.

Applied to the developing lens of the 14-day-old chick embryo, in organ culture conditions, chloroquine prevented the elongation of the primary lens fibres, destroyed the equatorial ones and provoked vacuolisation and/or destruction in the epithelial cells.