SP-A1 and SP-A2 variants differentially enhance association ofPseudomonas aeruginosawith rat alveolar macrophages

2005 ◽  
Vol 288 (1) ◽  
pp. L150-L158 ◽  
Author(s):  
Anatoly N. Mikerov ◽  
Todd M. Umstead ◽  
Weixiong Huang ◽  
Wenlei Liu ◽  
David S. Phelps ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Single Gene ◽  
Protein A ◽  
Surfactant Protein ◽  
Phagocytic Index ◽  
Dependent Manner ◽  
Gene Products ◽  
Concentration Dependent Manner ◽  
Cell Association

Chronic airway inflammation caused by Pseudomonas aeruginosa is an important feature of cystic fibrosis (CF). Surfactant protein A (SP-A) enhances phagocytosis of P. aeruginosa. Two genes, SP-A1 and SP-A2, encode human SP-A. We hypothesized that genetically determined differences in the activity of SP-A1 and SP-A2 gene products exist. To test this, we studied association of a nonmucoid P. aeruginosa strain (ATCC 39018) with rat alveolar macrophages in the presence or absence of insect cell-expressed human SP-A variants. We used two trios, each consisting of SP-A1, SP-A2, and their coexpressed SP-A1/SP-A2 variants. We tested the 6A2and 6A4alleles (for SP-A1), the 1A0and 1A alleles (for SP-A2), and their respective coexpressed SP-A1/SP-A2 gene products. After incubation of alveolar macrophages with P. aeruginosa in the presence of the SP-A variants at 37°C for 1 h, the cell association of bacteria was assessed by light microscopy analysis. We found 1) depending on SP-A concentration and variant, SP-A2 variants significantly increased the cell association more than the SP-A1 variants (the phagocytic index for SP-A1 was ∼52–95% of the SP-A2 activity); 2) coexpressed variants at certain concentrations were more active than single gene products; and 3) the phagocytic index for SP-A variants was ∼18–41% of the human SP-A from bronchoalveolar lavage. We conclude that human SP-A variants in vitro enhance association of P. aeruginosa with rat alveolar macrophages differentially and in a concentration-dependent manner, with SP-A2 variants having a higher activity compared with SP-A1 variants.

scholarly journals Rat surfactant protein D enhances the production of oxygen radicals by rat alveolar macrophages

1992 ◽  
Vol 286 (1) ◽  
pp. 5-8 ◽  
Author(s):  
J F Van Iwaarden ◽  
H Shimizu ◽  
P H M Van Golde ◽  
D R Voelker ◽  
L M G Van Golde
Keyword(s):  
Alveolar Macrophages ◽  
Oxygen Radicals ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Surfactant Protein D ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Oxygen Radical Production ◽  
Stimulation Of

Rat surfactant protein D (SP-D) was shown to enhance the production of oxygen radicals by rat alveolar macrophages. This enhancement, which was determined by a lucigenin-dependent chemiluminescence assay, was maximal after 18 min at an SP-D concentration of 0.2 micrograms/ml. Surfactant lipids did not influence the stimulation of alveolar macrophages by SP-D, whereas the oxygen-radical production of these cells induced by surfactant protein A was inhibited by the lipids in a concentration-dependent manner.

Role of clathrin-mediated endocytosis of surfactant protein A by alveolar macrophages in intracellular signaling

2009 ◽  
Vol 296 (3) ◽  
pp. L430-L441 ◽  
Author(s):  
Christina Moulakakis ◽  
Cordula Stamme
Keyword(s):  
Alveolar Macrophages ◽  
Intracellular Signaling ◽  
Protein A ◽  
Surfactant Protein ◽  
Kinase Assay ◽  
Coated Vesicle ◽  
Dependent Manner ◽  
Tnf Α

We recently provided evidence that anti-inflammatory macrophage activation, i.e., the inhibition of constitutive and signal-induced NF-κB activity by the pulmonary collectin surfactant protein (SP)-A, critically involves a promoted stabilization of IκB-α, the predominant inhibitor of NF-κB, via posttranscriptional mechanisms comprising the activation of atypical (a)PKCζ. SP-A uptake and degradation by alveolar macrophages (AMφ) occur in a receptor-mediated, clathrin-dependent manner. However, a mutual link between endocytosis of and signaling by SP-A remains elusive. The aim of this study was to investigate whether clathrin-mediated endocytosis (CME) of SP-A by AMφ is a prerequisite for its modulation of the IκB-α/NF-κB pathway. The inhibition of clathrin-coated pit (CCP) formation and clathrin-coated vesicle (CCV) formation/budding abrogates SP-A-mediated IκB-α stabilization and SP-A-mediated inhibition of LPS-induced NF-κB activation in freshly isolated rat AMφ, as determined by Western analysis, fluorescence-activated cell sorting, confocal microscopy, and EMSA. Actin depolymerization and inhibition of CCP formation further abolished SP-A-mediated inhibition of LPS-induced TNF-α release, as determined by ELISA. In addition, SP-A-induced atypical PKCζ activation was abolished by pretreatment of AMφ with CCV inhibitors as determined by in vitro immunocomplex kinase assay. Although CME is classically considered as a means to terminate signaling, our results demonstrate that SP-A uptake via CME by AMφ has to precede the initiation of SP-A signaling.

Surfactant protein A enhances the binding and deacylation ofE. coliLPS by alveolar macrophages

1999 ◽  
Vol 276 (3) ◽  
pp. L540-L547 ◽  
Author(s):  
Cordula Stamme ◽  
Jo Rae Wright
Keyword(s):  
Alveolar Macrophages ◽  
Protein A ◽  
Surfactant Protein ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cell Surface Molecule ◽  
Molar Ratios ◽  
A Cell ◽  
First Time ◽  
Lps Binding

Surfactant protein (SP) A and SP-D are involved in multiple immunomodulatory functions of innate host defense partly via their interaction with alveolar macrophages (AMs). In addition, both SP-A and SP-D bind to bacterial lipopolysaccharide (LPS). To investigate the functional significance of this interaction, we first tested the ability of SP-A and SP-D to enhance the binding of tritium-labeled Escherichia coli LPS to AMs. In contrast to SP-D, SP-A enhanced the binding of LPS by AMs in a time-, temperature-, and concentration-dependent manner. Coincubation with surfactant-like lipids did not affect the SP-A-mediated enhancement of LPS binding. At SP-A-to-LPS molar ratios of 1:2–1:3, the LPS binding by AMs reached 270% of control values. Second, we investigated the role of SP-A in regulating the degradation of LPS by AMs. In the presence of SP-A, deacylation of LPS by AMs increased by ∼2.3-fold. Pretreatment of AMs with phosphatidylinositol-specific phospholipase C had no effect on the SP-A-enhanced LPS binding but did reduce the amount of serum-enhanced LPS binding by 50%, suggesting that a cell surface molecule distinct from CD14 mediates the effect of SP-A. Together the results for the first time provide direct evidence that SP-A enhances LPS binding and degradation by AMs.

scholarly journals Impact of ozone exposure on the phagocytic activity of human surfactant protein A (SP-A) and SP-A variants

2008 ◽  
Vol 294 (1) ◽  
pp. L121-L130 ◽  
Author(s):  
Anatoly N. Mikerov ◽  
Todd M. Umstead ◽  
Xiaozhuang Gan ◽  
Weixiong Huang ◽  
Xiaoxuan Guo ◽  
...  
Keyword(s):  
Phagocytic Activity ◽  
Protein A ◽  
Surfactant Protein ◽  
Redox Status ◽  
Ozone Exposure ◽  
Surfactant Protein A ◽  
Phagocytic Index ◽  
Dependent Manner

Surfactant protein A (SP-A) enhances phagocytosis of Pseudomonas aeruginosa. SP-A1 and SP-A2 encode human (h) SP-A; SP-A2 products enhance phagocytosis more than SP-A1. Oxidation can affect SP-A function. We hypothesized that in vivo and in vitro ozone-induced oxidation of SP-A (as assessed by its carbonylation level) negatively affects its function in phagocytosis (as assessed by bacteria cell association). To test this, we used P. aeruginosa, rat alveolar macrophages (AMs), hSP-As with varying levels of in vivo (natural) oxidation, and ozone-exposed SP-A2 (1A, 1A0) and SP-A1 (6A2, 6A4) variants. SP-A oxidation levels (carbonylation) were measured; AMs were incubated with bacteria in the presence of SP-A, and the phagocytic index was calculated. We found: 1) the phagocytic activity of hSP-A is reduced with increasing levels of in vivo SP-A carbonylation; 2) in vitro ozone exposure of hSP-A decreases its function in a dose-dependent manner as well as its ability to enhance phagocytosis of either gram-negative or gram-positive bacteria; 3) the activity of both SP-A1 and SP-A2 decreases in response to in vitro ozone exposure of proteins with SP-A2 being affected more than SP-A1. We conclude that both in vivo and in vitro oxidative modifications of SP-A by carbonylation reduce its ability to enhance phagocytosis of bacteria and that the activity of SP-A2 is affected more by in vitro ozone-induced oxidation. We speculate that functional differences between SP-A1 and SP-A2 exist in vivo and that the redox status of the lung microenvironment differentially affects function of SP-A1 and SP-A2.

Oxygen modulates the differentiation of human fetal lung in vitro and its responsiveness to cAMP

1993 ◽  
Vol 264 (5) ◽  
pp. L465-L474 ◽  
Author(s):  
M. J. Acarregui ◽  
J. M. Snyder ◽  
C. R. Mendelson
Keyword(s):  
Gene Expression ◽  
Atmospheric Oxygen ◽  
Protein A ◽  
Morphological Differentiation ◽  
Fetal Lung ◽  
Morphological Development ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Human Fetal Lung

Previously, it was found that lung explants from mid-trimester human abortuses differentiate spontaneously in organ culture in serum-free defined medium in an atmosphere of 95% air-5% CO2. Dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) treatment of human fetal lung in culture increases the rate of morphological differentiation and enhances expression of the surfactant protein A (SP-A) gene. To begin to define the factors responsible for this accelerated in vitro differentiation, we analyzed the effects of atmospheric oxygen on the morphological and biochemical development of human fetal lung in culture and on responsiveness of the cultured tissue to DBcAMP. We found that when lung explants were maintained in an atmosphere containing 1% oxygen they failed to differentiate spontaneously and no induction of SP-A gene expression was apparent. Furthermore, at 1% oxygen, DBcAMP had no effect to stimulate morphological differentiation or SP-A gene expression. When lung tissues that had been maintained for 5 days in 1% oxygen were transferred to an environment containing 20% oxygen, there was rapid morphological development and induction of SP-A gene expression. The effects on morphological development were manifest within 24 h of transfer to the 20% oxygen environment; within 72 h, a marked stimulatory effect of DBcAMP on SP-A gene expression also was observed. Our findings further suggest that the effects of oxygen on the levels of SP-A and SP-A mRNA are concentration dependent. Interestingly, the inductive effects of DBcAMP on SP-A gene expression were apparent only at oxygen concentrations > or = 10%. Morphological differentiation of the cultured human fetal lung tissue also was influenced by oxygen in a concentration-dependent manner. These findings suggest that oxygen plays an important permissive role in the spontaneous differentiation of human fetal lung in vitro.

Degradation of surfactant lipids and surfactant protein A by alveolar macrophages in vitro

1995 ◽  
Vol 268 (5) ◽  
pp. L772-L780 ◽  
Author(s):  
J. R. Wright ◽  
D. C. Youmans
Keyword(s):  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Alveolar Type ◽  
Dependent Manner ◽  
Type Ii ◽  
Alveolar Type Ii Cell ◽  
Degradation Rates ◽  
Type Ii Cell

Pulmonary surfactant is synthesized and secreted into the airspaces by the alveolar type II cell. After it is secreted, surfactant undergoes a series of poorly understood transformations resulting in formation of a surface tension-reducing surface at the air-liquid interface. The by-products of the surface film and/or other products of surfactant metabolism are eventually cleared from the alveolar space. Both the alveolar type II cell and the macrophage are thought to be involved in surfactant clearance and have been shown to internalize surfactant lipid in vitro. The goal of the current investigation was to characterize further and to quantitate the role of the macrophage in surfactant clearance by investigating the uptake and metabolism of surfactant lipids and surfactant protein A (SP-A) by macrophages in vitro. SP-A enhanced the uptake of lipids by macrophages in a time-, temperature-, and concentration-dependent manner. In contrast, neither of the collagen-like proteins SP-D or C1q enhanced the uptake. Phosphatidylcholine was rapidly degraded by macrophages and the degradation occurred both in the presence and absence of SP-A. In addition, macrophages degrade SP-A by a process that is time- and temperature-dependent. These results and calculations of uptake and degradation rates suggest that macrophages may contribute significantly to the process of surfactant clearance.

Human SP-A protein variants derived from one or both genes stimulate TNF-α production in the THP-1 cell line

2000 ◽  
Vol 278 (5) ◽  
pp. L946-L954 ◽  
Author(s):  
Guirong Wang ◽  
David S. Phelps ◽  
Todd M. Umstead ◽  
Joanna Floros
Keyword(s):  
Tumor Necrosis ◽  
Single Gene ◽  
Surfactant Protein ◽  
Functional Genes ◽  
Gene Products ◽  
Functional Differences ◽  
Tnf Α ◽  
Protein Variants ◽  
Necrosis Factor

In humans, two functional genes of surfactant protein (SP) A, SP-A1 and SP-A2, and several alleles of each functional gene have been characterized. SP-A is a multimeric molecule consisting of six trimers. Each trimer contains two SP-A1 molecules and one SP-A2 molecule. Until now, it has been unclear whether a single SP-Agene product is functional or whether there are functional differences either among alleles or between single-gene SP-A products and SP-A products derived from both genes. We tested the ability of in vitro expressed SP-A variants to stimulate tumor necrosis factor (TNF)-α production by THP-1 cells. We observed that 1) single-gene products and products derived from both genes stimulate TNF-α production, 2) there are differences among SP-A1 and SP-A2 alleles in their ability to stimulate TNF-α production, and 3) the increases in TNF-α production are lower after treatment with the SP-A1 alleles than after treatment with the SP-A2 alleles. Furthermore, coexpressed SP-As from SP-A1 and SP-A2 genes have a higher activity compared with SP-As from individual alleles or mixed SP-As from SP-A1and SP-A2 genes. These data suggest that the SP-A-induced increases in TNF-α levels differ among SP-A variants and appear to be affected by SP-A genotype and whether SP-A is derived from one or both genes.

scholarly journals Human surfactant protein A with two distinct oligomeric structures which exhibit different capacities to interact with alveolar type II cells

1996 ◽  
Vol 317 (3) ◽  
pp. 939-944 ◽  
Author(s):  
Akiko HATTORI ◽  
Yoshio KUROKI ◽  
Hitoshi SOHMA ◽  
Yoshinori OGASAWARA ◽  
Toyoaki AKINO
Keyword(s):  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Dependent Manner ◽  
Type Ii ◽  
Concentration Dependent Manner ◽  
Lower Affinity ◽  
Alveolar Proteinosis

The lung lavage fluids from patients with pulmonary alveolar proteinosis have been generally used as a source for human surfactant protein A (SP-A). We have recently found that a multimerized form of SP-A oligomer (alveolar proteinosis protein-I, APP-I) exists besides the normal-sized octadecamer (APP-II) in SP-As isolated from the patients. When analysed by Bio-Gel A15m column chromatography in 5 mM Tris buffer (pH 7.4), the apparent molecular masses of APP-I and APP-II were 1.65 MDa and 0.93 MDa, respectively. Gel-filtration analysis also revealed that APP-II is clearly separated from APP-I in the presence of 2 mM Ca2+ and 150 mM NaCl. We investigated the abilities of both SP-A oligomers to regulate phospholipid secretion and to bind to alveolar type II cells. Although APP-I inhibited lipid secretion, it was clearly a less effective inhibitor than APP-II. IC50 for inhibition of lipid secretion was apparently 0.23±0.08 µg/ml (0.14±0.05 nM) and 0.055±0.019 µg/ml (0.059±0.020 nM) for APP-I and APP-II, respectively. Both proteins bound to monolayers of type II cells in a concentration-dependent manner; however, APP-I clearly had a lower affinity to bind to type II cells. The apparent dissociation contants were, Kd = 2.31±0.70 µg/ml (1.40±0.43 nM) and 0.89±0.22 µg/ml (0.95±0.24 nM) for APP-I and APP-II, respectively. Excess unlabelled rat SP-A replaced 45% of 125I-APP-I and 77% of 125I-APP-II for type II cell binding. Although 125I-APP-II competed with excess unlabelled APP-I or APP-II, 125I-APP-I failed to compete and instead its binding rather increased in the presence of unlabelled APPs. The biotinylated APP-I bound to APP-I and APP-II coated on to microtitre wells in a concentration-dependent manner, indicating that APP-I interacts with APPs. This study demonstrates that the multimerized form of human SP-A oligomer exhibits the following attributes: (1) the reduced capacity to regulate phospholipid secretion from type II cells, and (2) lower affinity to bind to type II cells, and that the integrity of a flower-bouquet-like octadecameric structure of SP-A oligomer is important for the expression of full activity of this protein, indicating the importance of the oligomeric structure of mammalian lectins with collagenous domains.

scholarly journals Titanium dioxide nanoparticles induce in vitro autophagy

2018 ◽  
Vol 38 (1) ◽  
pp. 56-64 ◽  
Author(s):  
X Dai ◽  
R Liu ◽  
N Li ◽  
J Yi
Keyword(s):  
Titanium Dioxide ◽  
Messenger Rna ◽  
Titanium Dioxide Nanoparticles ◽  
Beclin 1 ◽  
Raw 264.7 Cells ◽  
Phagocytic Index ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Concentration Dependent Manner

Aim: Concerns about the possible toxicity to environment and human health of titanium dioxide nanoparticles (TiO2 NPs) are increasing. The aim of this study was to investigate the relationship between toxicology and autophage in vitro. Methods: RAW 264.7 cells were exposed to five concentrations (50, 100, 200, 300, and 400 μg/mL) and two particle size of TiO2 NPs (30 and 100 nm) for 24 h. Results: The results showed that TiO2 NPs decreased cell viability, phagocytic rate, and phagocytic index in a concentration-dependent manner, thereby inducing autophagy. TiO2 NPs-induced autophagy was indicated by monodansyl cadaverine staining and transmission electron microscopy. TiO2 NPs-induced messenger RNA expression of autophagy-related proteins LC3 and Beclin-1 was also significantly increased compared with those of the unexposed control cells. LC3 and Beclin-1 protein expression levels were markedly increased with the increase of TiO2 NPs concentrations. Conclusion: These results suggest the possibility that TiO2 NPs-induced toxicology probably plays a key role in autophagy in RAW 264.7 cells, and further exhaustive research on the harmful effects of these NPs in relevant organisms is needed for their safe application.

Pulmonary surfactant protein A stimulates chemotaxis of alveolar macrophage

1993 ◽  
Vol 264 (4) ◽  
pp. L338-L344 ◽  
Author(s):  
J. R. Wright ◽  
D. C. Youmans
Keyword(s):  
Pulmonary Surfactant ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Macrophage Migration ◽  
Dependent Manner ◽  
Directed Movement ◽  
Concentration Dependent Manner ◽  
Pulmonary Surfactant Protein ◽  
Checker Board

Pulmonary surfactant modulates several functions of alveolar macrophages including phagocytosis, killing, and chemotaxis. We hypothesized that the reported stimulatory effect of surfactant on macrophage migration was mediated by one of the surfactant proteins, SP-A. We found that macrophage migration was stimulated by SP-A in a concentration-dependent manner. A concentration of 105 micrograms SP-A/ml enhanced migration approximately 10-fold. Heat treatment or reduction and alkylation of SP-A reduced its stimulatory effect. A checker-board analysis showed that SP-A stimulated migration primarily by enhancing chemotaxis (directed movement) rather than chemokinesis (random movement). The interaction of SP-A with macrophages may be mediated at least partly by the collagen-like domain of SP-A. We speculate that SP-A may play a multifunctional role in regulating pulmonary immune response by stimulating multiple macrophage functions.

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