Specific binding of surfactant apoprotein SP-A to rat alveolar macrophages

1992 ◽  
Vol 262 (4) ◽  
pp. L412-L417 ◽  
Author(s):  
U. Pison ◽  
J. R. Wright ◽  
S. Hawgood
Keyword(s):  
Alveolar Macrophages ◽  
Specific Binding ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Dose Dependent Fashion ◽  
Type V ◽  
Dose Dependent ◽  
Surfactant Apoprotein

Surfactant protein A (SP-A) influences the function of alveolar macrophages in vitro. In this study the characteristics of the binding of 125I-labeled SP-A to rat alveolar macrophages has been investigated. The binding of SP-A to alveolar macrophages at 4 degrees C was saturable with half-maximal binding at a SP-A concentration of 4 micrograms/ml. Bound SP-A was rapidly displaced by an excess of unlabeled SP-A. The binding of labeled SP-A to the alveolar macrophages was blocked in a dose-dependent fashion by unlabeled SP-A, the collagen-like protein C1q and type V collagen but not by bovine serum albumin. These results suggest that a component of the interaction between SP-A and alveolar macrophages is mediated through the collagen-like domain of SP-A and that the characteristics of this interaction are consistent with there being a specific receptor for SP-A on the surface of alveolar macrophages.

Carbon dioxide enhances nitration of surfactant protein A by activated alveolar macrophages

2000 ◽  
Vol 278 (5) ◽  
pp. L1025-L1031 ◽  
Author(s):  
Sha Zhu ◽  
Khaled F. Basiouny ◽  
John P. Crow ◽  
Sadis Matalon
Keyword(s):  
Nitric Oxide ◽  
Alveolar Macrophages ◽  
Protein A ◽  
Surfactant Protein ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Dose Dependent Fashion ◽  
Carbonyl Formation ◽  
Oxide Synthase ◽  
Dose Dependent

We assessed whether reactive oxygen-nitrogen intermediates generated by alveolar macrophages (AMs) oxidized and nitrated human surfactant protein (SP) A. SP-A was exposed to lipopolysaccharide (100 ng/ml)-activated AMs in 15 mM HEPES (pH 7.4) for 30 min in the presence and absence of 1.2 mM CO2. In the presence of CO2, lipopolysaccharide-stimulated AMs had significantly higher nitric oxide synthase (NOS) activity (as quantified by the conversion ofl-[U-14C]arginine tol-[U-14C]citrulline) and secreted threefold higher levels of nitrate plus nitrite in the medium [28 ± 3 vs. 6 ± 1 (SE) nmol ⋅ 6.5 h−1 ⋅ 106AMs−1]. Western blotting studies of immunoprecipitated SP-A indicated that CO2 enhanced SP-A nitration by AMs and decreased carbonyl formation. CO2(0–1.2 mM) also augmented peroxynitrite (0.5 mM)-induced SP-A nitration in a dose-dependent fashion. Peroxynitrite decreased the ability of SP-A to aggregate lipids, and this inhibition was augmented by 1.2 mM CO2. Mass spectrometry analysis of chymotryptic fragments of peroxynitrite-exposed SP-A showed nitration of two tyrosines (Tyr164 and Tyr166) in the absence of CO2 and three tyrosines (Tyr164, Tyr166, and Tyr161) in the presence of 1.2 mM CO2. These findings indicate that physiological levels of peroxynitrite, produced by activated AMs, nitrate SP-A and that CO2 increased nitration, at least partially, by enhancing enzymatic nitric oxide production.

Lung surfactant protein A (SP-A) activates a phosphoinositide/calcium signaling pathway in alveolar macrophages

1995 ◽  
Vol 108 (12) ◽  
pp. 3695-3702
Author(s):  
D. Ohmer-Schrock ◽  
C. Schlatterer ◽  
H. Plattner ◽  
J. Schlepper-Schafer
Keyword(s):  
Calcium Signaling ◽  
Signaling Pathway ◽  
Alveolar Macrophages ◽  
Lung Surfactant ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Calcium Signaling Pathway ◽  
Lung Surfactant Protein ◽  
Dose Dependent

Lung surfactant protein A (SP-A), the main protein component of lung surfactant which lines the alveoli, strongly enhances serum-independent phagocytosis of bacteria by rat alveolar macrophages. We tested if the effect of SP-A is due to interaction with the macrophages or to opsonization of the bacteria. In phagocytosis assays with fluorescein isothiocyanate labeled bacteria, SP-A had no opsonic effect on Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, but enhanced phagocytosis by acting only on the macrophages. We characterized this activation mechanism. With single cell measurements of fura-2 loaded cells we demonstrate that SP-A raises the intracellular free calcium ion concentration 6 to 8 seconds after addition. This calcium mobilization is dose-dependent in that increased SP-A concentrations lead to a higher percentage of responding cells. Additionally, SP-A leads to a dose-dependent and transient generation of inositol 1,4.5-trisphosphate. Release of intracellular stored calcium by SP-A is a prerequisite for its stimulatory effect on phagocytosis, since SP-A-induced enhancement of phagocytosis can be impaired by prior addition of thapsigargin, a Ca(2+)-ATPase inhibitor that leads to depletion of intracellular calcium stores. We conclude that SP-A activates a phosphoinositide/calcium signaling pathway in alveolar macrophages leading to enhanced serum-independent phagocytosis of bacteria.

scholarly journals A novel procedure for the rapid isolation of surfactant protein A with retention of its alveolar-macrophage-stimulating properties

1995 ◽  
Vol 309 (2) ◽  
pp. 551-555 ◽  
Author(s):  
J F van Iwaarden ◽  
F Teding van Berkhout ◽  
J A Whitsett ◽  
R S Oosting ◽  
L M G van Golde
Keyword(s):  
Alveolar Macrophage ◽  
Alveolar Macrophages ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Normal Rat ◽  
Normal Human ◽  
Novel Method ◽  
Alveolar Proteinosis ◽  
Simplex Virus

Previous studies have shown that surfactant protein A (SP-A) derived from alveolar-proteinosis patients activates rat alveolar macrophages. However, it is not known if normal rat, dog and human SP-A can also stimulate alveolar macrophages. As alveolar-proteinosis SP-A has a slightly different structure from ordinary SP-A, it would be possible that the ascribed alveolar-macrophage-stimulating properties of SP-A are restricted to alveolar-proteinosis SP-A. To clarify this issue, we isolated SP-A from normal rat and dog pulmonary surfactants, using the same isolation technique commonly used for the isolation of alveolar-proteinosis SP-A, i.e. by butanol precipitation. In contrast with human alveolar-proteinosis SP-A, rat and dog SP-A obtained thus could not activate rat alveolar macrophages to produce oxygen radicals or enhance the phagocytosis of fluorescein isothiocyanate-labelled herpes simplex virus. However, rat, dog and normal human SP-A isolated by a novel method, involving extraction from pulmonary surfactant by using n-octyl beta-D-glucopyranoside and subsequent purification by cation-exchange chromatography, were able to elicit an oxidative burst in rat as well as normal human alveolar macrophages. In addition, dog and rat SP-A obtained thus stimulated the phagocytosis of herpes simplex virus by rat alveolar macrophages. These findings indicate that normal human, rat and dog SP-A have the same alveolar-macrophage-stimulating properties as human alveolar proteinosis SP-A. Dog and rat SP-A isolated by this novel method had the same Ca(2+)-dependent self-aggregation and lipid-aggregation properties as SP-A isolated by butanol precipitation. The new and milder isolation procedure yielded SP-A of high purity, as judged by SDS/PAGE and ELISA.

scholarly journals Surfactant protein D binding to alveolar macrophages

1994 ◽  
Vol 300 (1) ◽  
pp. 237-242 ◽  
Author(s):  
K Miyamura ◽  
L E A Leigh ◽  
J Lu ◽  
J Hopkin ◽  
A López Bernal ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Serum Proteins ◽  
Specific Binding ◽  
Lectin Binding ◽  
Protein A ◽  
Direct Interaction ◽  
Surfactant Protein ◽  
Lung Epithelial Cells ◽  
Surfactant Protein D ◽  
Apparent Dissociation Constant

Surfactant protein D (SP-D) is a lung-specific protein, synthesized and secreted by lung epithelial cells. It belongs to group III of the family of C-type lectins; each member of this group has an unusual overall structure consisting of multiple globular ‘head’ regions (which contain the C-type lectin domains) linked by triple-helical, collagen-like, strands. This group includes the surfactant protein A (SP-A) and the serum proteins mannan-binding protein, conglutinin and collectin-43, all of which have been shown to bind to the C1q receptor found on a wide variety of cells, including macrophages. Both SP-D and SP-A have been shown to enhance oxygen radical production by alveolar macrophages. Although this strongly suggests a direct interaction between SP-D and a specific receptor on alveolar macrophages, it is still unclear whether SP-D binds to the same receptor used by SP-A and/or C1q. Human SP-D was isolated from amniotic fluid and was radiolabelled using 125I. Alveolar macrophages were isolated from human bronchioalveolar lavage fluid, and also from bovine lung washings, by differential adhesion to 24-well tissue-culture plates. The study was carried out using EDTA-containing buffers, to eliminate Ca(2+)-dependent C-type lectin binding, and was also carried out at 4 degrees C to eliminate possible internalization by the cells. 125I-SP-D showed specific binding to alveolar macrophages in both a time- and concentration-saturable manner. The binding was inhibited, by approx. 90%, on addition of a 200-fold excess of unlabelled SP-D. The apparent dissociation constant (Kd) was (3.6 +/- 1.3) x 10(-11) M, based on the assumption that native SP-D is assembled as a dodecamer of 12 identical polypeptides of 43 kDa to yield a protein of 516 kDa. C1q was also shown to bind alveolar macrophages (Kd 3 x 10(-6) M), but addition of C1q did not show inhibition of the binding of 125I-SP-D to the macrophages. We conclude that SP-D binds specifically to alveolar macrophages and the receptor involved is different from that utilized by C1q.

Surfactant protein A prevents silica-mediated toxicity to rat alveolar macrophages

2000 ◽  
Vol 278 (4) ◽  
pp. L713-L718 ◽  
Author(s):  
Robert W. Spech ◽  
Paul Wisniowski ◽  
Diane L. Kachel ◽  
Jo Rae Wright ◽  
William J. Martin
Keyword(s):  
Cell Injury ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Occupational Lung Disease ◽  
Direct Injury ◽  
Surfactant Apoprotein ◽  
Surfactant Apoprotein A ◽  
Cytotoxic Index

Silicosis is a serious occupational lung disease associated with irreversible pulmonary fibrosis. The interaction between inhaled crystalline silica and the alveolar macrophage (AM) is thought to be a key event in the development of silicosis and fibrosis. Silica can cause direct injury to AMs and can induce AMs to release various inflammatory mediators. Acute silicosis is also characterized by a marked elevation in surfactant apoprotein A (SP-A); however, the role of SP-A in silicosis is unknown. We investigated whether SP-A directly affects the response of AMs to silica. In this study, the degree of silica toxicity to cultured rat AMs as assessed by a 51Cr cytotoxicity assay was shown to be dependent on the time of exposure and the concentration and size of the silica particles. Silica directly injured rat AMs as evidenced by a cytotoxic index of 32.9 ± 2.5, whereas the addition of rat SP-A (5 μg/ml) significantly reduced the cytotoxic index to 16.6 ± 1.2 ( P < 0.001). This effect was reversed when SP-A was incubated with either polyclonal rabbit anti-rat SP-A antibody ord-mannose. These data indicate that SP-A mitigates the effect of silica on AM viability, and this effect may involve the carbohydrate recognition domain of SP-A. The elevation of SP-A in acute silicosis may serve as a normal host response to prevent lung cell injury after exposure to silica.

Exposure of surfactant protein A to ozone in vitro and in vivo impairs its interactions with alveolar cells

1992 ◽  
Vol 262 (1) ◽  
pp. L63-L68 ◽  
Author(s):  
R. S. Oosting ◽  
J. F. Van Iwaarden ◽  
L. Van Bree ◽  
J. Verhoef ◽  
L. M. Van Golde ◽  
...  
Keyword(s):  
Superoxide Anion ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Alveolar Type ◽  
Type Ii ◽  
Superoxide Anion Production ◽  
Anion Production

This study focused on the question of whether exposure of surfactant protein A (SP-A) to ozone affected properties of this protein that may be involved in regulating alveolar type II cell and alveolar macrophage functions. In vitro exposure of human or canine SP-A to ozone reduced the ability of this protein to inhibit phorbol-ester induced secretion of [3H]phosphatidylcholine by alveolar type II cells in culture. Ozone-exposed human SP-A showed a decreased ability to enhance phagocytosis of herpes simplex virus and to stimulate superoxide anion production by alveolar macrophages. Experiments with elastase showed that ozone-exposed canine SP-A was more susceptible to proteolysis. A conformational change of the protein could underlie this phenomenon. Surfactant isolated from ozone-exposed rats (0.4 ppm ozone for 12 h) was also less able to stimulate superoxide anion production by alveolar macrophages than surfactant from control rats, which suggested that SP-A in vivo was also susceptible to ozone. The results of this study suggest that SP-A-alveolar cell interactions can be inhibited by ozone exposure, which may contribute to the toxicity of ozone in the lungs.

scholarly journals Effects of endotoxin on surfactant protein A and D stimulation of NO production by alveolar macrophages

1999 ◽  
Vol 276 (4) ◽  
pp. L650-L658 ◽  
Author(s):  
Jo Rae Wright ◽  
Daniel F. Zlogar ◽  
Julie C. Taylor ◽  
Thomas M. Zlogar ◽  
Clara I. Restrepo
Keyword(s):  
Alveolar Macrophages ◽  
Protein A ◽  
Surfactant Protein ◽  
Optimal Recovery ◽  
Surfactant Protein A ◽  
No Production ◽  
Nitrite Production ◽  
Endotoxin Contamination ◽  
Nitric Oxide Metabolites ◽  
Endotoxin Content

Surfactant protein (SP) A and SP-D affect numerous functions of immune cells including enhancing phagocytosis of bacteria and production of reactive species. Previous studies have shown that SP-A and SP-D bind to a variety of bacteria and to the lipopolysaccharide (LPS) components of their cell walls. In addition, purified preparations of SPs often contain endotoxin. The goals of this study were 1) to evaluate the effects of SP-A and SP-D and complexes of SPs and LPS on the production of nitric oxide metabolites by rat alveolar macrophages and 2) to evaluate methods for the removal of endotoxin with optimal recovery of SP. Incubation of SP-A or SP-D with polymyxin, 100 mM N-octyl-β-d-glucopyranoside, and 2 mM EDTA followed by dialysis was the most effective method of those tested for reducing endotoxin levels. Commonly used storage buffers for SP-D, but not for SP-A, inhibited the detection of endotoxin. There was a correlation between the endotoxin content of the SP-A and SP-D preparations and their ability to stimulate production of nitrite by alveolar macrophages. SP-A and SP-D treated as described above to remove endotoxin did not stimulate nitrite production. These studies suggest that the functions of SP-A and SP-D are affected by endotoxin and illustrate the importance of monitoring SP preparations for endotoxin contamination.

Effects of surfactant protein A and surfactant lipids on lymphocyte proliferation in vitro

1994 ◽  
Vol 267 (4) ◽  
pp. L357-L364 ◽  
Author(s):  
S. G. Kremlev ◽  
T. M. Umstead ◽  
D. S. Phelps
Keyword(s):  
Lymphocyte Proliferation ◽  
Cell Function ◽  
Immune Cell ◽  
Pulmonary Inflammation ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Inhibitory Influence ◽  
Surfactant Replacement Therapy

We studied the effects of dipalmitoyl L-alpha-phosphatidylcholine (DPPC), Survanta, surfactant protein A (SP-A), and mixtures of these substances on mitogen-induced lymphocyte proliferation using concanavalin A as a mitogen. A concentration-dependent suppression of proliferation was observed with 50-250 micrograms/ml of DPPC or Survanta. However, when SP-A was added to cultures, proliferation was stimulated. The inhibitory effects of DPPC and Survanta were altered in mixtures that contained SP-A. When added to 50 micrograms/ml of Survanta, SP-A reversed the inhibitory influence of Survanta and caused increased proliferation. These findings suggest that surfactant phospholipids cause a suppression of mitogen-induced lymphocyte proliferation, which is reversed somewhat by addition of SP-A. We hypothesize that immune cell function in the lung varies with changes in the relative amounts of surfactant components. Changes in surfactant composition may occur during pulmonary inflammation or infection or with surfactant replacement therapy and may influence immune and inflammatory processes in the lung.

Endocytic pathway for surfactant protein A in human macrophages: binding, clathrin-mediated uptake, and trafficking through the endolysosomal pathway

2006 ◽  
Vol 290 (2) ◽  
pp. L334-L342 ◽  
Author(s):  
Joy E. Crowther ◽  
Larry S. Schlesinger
Keyword(s):  
Specific Binding ◽  
Lung Surfactant ◽  
Protein A ◽  
Surfactant Protein ◽  
Endocytic Pathway ◽  
Surfactant Protein A ◽  
Coated Pits ◽  
Protein Protein Interaction ◽  
Intracellular Compartments ◽  
Over Time

In the noninflamed lung, surfactant protein A (SP-A) acts as an anti-inflammatory molecule through its effects on macrophage (MΦ) function, modulating cytokine and reactive oxygen and nitrogen intermediate production. The receptors responsible for these effects of SP-A on human MΦ are not clear, although SP-A binding to several proteins has been described. In this study, we demonstrate high-affinity specific binding of SP-A to primary human MΦ. SP-A binding was inhibited by EGTA, indicating calcium dependence. However, mannan did not inhibit SP-A binding, suggesting that binding is mediated by a direct protein-protein interaction that does not involve carbohydrate recognition. Our laboratory has previously shown that SP-A is rapidly endocytosed by human MΦ into discrete vesicles. Although previous work indicates that SP-A is ultimately degraded by murine MΦ over time, the trafficking pathway of SP-A through MΦ after uptake has not been reported and is of potential biological importance. We examined trafficking of SP-A in human MΦ by electron and confocal microscopy and show for the first time that SP-A is endocytosed by primary human MΦ through clathrin-coated pits and colocalizes sequentially over time with the early endosome marker EEA1, late endosome marker lamp-1, and lysosome marker cathepsin D. We conclude that SP-A binds to receptor(s) on human MΦ, is endocytosed by a receptor-mediated, clathrin-dependent process, and trafficks through the endolysosomal pathway. These studies provide further insight into the interactions of SP-A with the MΦ cell surface and intracellular compartments that play important roles in SP-A modulation of lung MΦ biology.

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