C57BL/6 and BALB/c bronchoalveolar macrophages respond differently to exercise.

Macrophages from prototypical Th1 strains (e.g., C57BL/6) and Th2 strains (e.g., BALB/c) are classified as M-1 and M-2 phenotypes. We investigated the different phagocytic responses between M-1 and M-2 bronchoalveolar macrophages (BAMs) under resting and two various exercise conditions. At rest, M-1 BAMs showed higher phagocytic capacity of unopsonized particles, higher expression of MARCO (macrophage receptor with collagenous structure), and higher generation of NO than M-2 BAMs. Severe exercise, but not moderate exercise, significantly enhanced both phagocytosis of unopsonized particles and expression of MARCO in M-2 BAMs. In contrast, M-1 BAMs were unaffected by either exercise protocol. The phagocytosis of unopsonized particles was largely mediated by MARCO, especially in M-1 BAMs. Secreted products from cultured M-2 BAMs isolated after severe exercise, but not those from M-1 BAMs, enhanced BAM phagocytosis. The cultured M-1 BAMs secreted phagocytosis inhibitors, and this effect could be blocked by NO antagonists. Moreover, the extent of phagocytosis suppression induced by M-1 BAM-secreted products correlated with their production of nitrite/nitrate. Exogenous NO donors as well as NO derivatives, nitrite and nitrate, suppressed the BAM phagocytosis. We propose that while the severe exercise-enhanced phagocytosis in M-2 BAMs was largely mediated by MARCO up-regulation and secretion of stimulators, the lack of exercise effect in M-1 BAMs could be partially due to the constitutive secretion of NO-related suppressors. In conclusion, genetically different mice use different strategies in regulating BAM activity under resting conditions and in response to various exercise paradigms.     

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.     

Lung surfactant protein A (SP-A) enhances serum-independent phagocytosis of bacteria by alveolar macrophages.

Surfactant protein A (SP-A) is the main protein component of lung surfactant. We studied the involvement of SP-A in body defense, i.e., effect of SP-A on the phagocytosis of bacteria by alveolar macrophages. We show here that SP-A enhances the phagocytosis of some non-opsonized bacteria: Escherichia coli growing logarithmically (E. coli/log), Pseudomonas aeruginosa/log as well as from stationary phase (P. aeruginosa/stat) and Staphylococcus aureus/log. Furthermore, not only serum-independent phagocytosis was effected by SP-A but also phagocytosis of serum-opsonized S. aureus/stat. No effect of SP-A on phagocytosis was observed with E. coli/stat neither on serum-independent nor on serum-dependent phagocytosis and on phagocytosis of non-opsonized S. aureus/stat. Thus, effect of SP-A on phagocytosis is dependent on bacterial species and on the growth phase of the microorganisms, and this effect is concentration dependent. We studied two different human recombinant SP-As and SP-A isolated from lung lavage material from proteinosis patients. These SP-A molecules contain different isomeric chains, and they differ in complexity of their structure. Qualitatively, we found the same effect with all three substances. Quantitatively, the proteinosis SP-A that forms the most complex structure was the most effective. Taken together, we demonstrated a stimulating effect of SP-A on serum-independent as well as on serum-dependent phagocytosis of bacteria by alveolar macrophages, both depending on species and growth phase of the bacteria.     

Surfactant protein A regulates IgG-mediated phagocytosis in inflammatory neutrophils

Surfactant proteins (SP)-A and SP-D have been shown to affect the functions of a variety of innate immune cells and to interact with various immune proteins such as complement and immunoglobulins. The goal of the current study is to test the hypothesis that SP-A regulates IgG-mediated phagocytosis by neutrophils, which are major effector cells of the innate immune response that remove invading pathogens by phagocytosis and by extracellular killing mediated by reactive oxygen and nitrogen. We have previously shown that SP-A stimulates chemotaxis by inflammatory, but not peripheral, neutrophils. To evaluate the ability of SP-A to modulate IgG-mediated phagocytosis, polystyrene beads were coated with BSA and treated with anti-BSA IgG. SP-A significantly and specifically enhanced IgG-mediated phagocytosis by inflammatory neutrophils, but it had no effect on beads not treated with IgG. SP-A bound to IgG-coated beads and enhanced their uptake via direct interactions with the beads as well as direct interactions with the neutrophils. SP-A did not affect reactive oxygen production or binding of IgG to neutrophils and had modest effects on polymerization of actin. These data suggest that SP-A plays an important role in mediating the phagocytic response of neutrophils to IgG-opsonized particles.     

The role of pulmonary collectin N-terminal domains in surfactant structure,function, and homeostasis in vivo

The N-terminal domains of the lung collectins, surfactant proteins A (SP-A) and D (SP-D), are critical for surfactant phospholipid interactions and surfactant homeostasis, respectively. To further assess the importance of lung collectin N-terminal domains in surfactant structure and function, a chimeric SP-D/SP-A (D/A) gene was constructed by substituting nucleotides encoding amino acids Asn(1)-Ala(7) of rat SP-A with the corresponding N-terminal sequences from rat SP-D, Ala(1)-Asn(25). Recombinant D/A migrated as a 35-kDa band on reducing SDS-PAGE and as a ladder of disulfide-linked multimers under nonreducing conditions. The recombinant D/A bound and aggregated phosphatidylcholine containing vesicles as effectively as rat SP-A. Mice in which endogenous pulmonary collectins were replaced with D/A were developed by human SP-C promoter-driven overexpression of the D/A gene in SP-A(-/-) and SP-D(-/-) animals. Analysis of lavage fluid from SP-A(-/-,D/A) mice revealed that glycosylated, oligomeric D/A was secreted into the air spaces at levels that were comparable with the authentic collectins and that the N-terminal interchange converted SP-A from a "bouquet" to a cruciform configuration. Transmission electron microscopy of surfactant from the SP-A(-/-,D/A) mice revealed atypical tubular myelin containing central "target-like" electron density. Surfactant isolated from SP-A(-/-,D/A) mice exhibited elevated surface tension both in the presence and absence of plasma inhibitors, but whole lung compliance of the SP-A(-/-,D/A) animals was not different from the SP-A(-/-) littermates. Lung-specific overexpression of D/A in the SPD(-/-) mouse resulted in hetero-oligomer formation with mouse SP-A and did not correct the air space dilation or phospholipidosis that occurs in the absence of SP-D. These studies indicate that the N terminus of SP-D 1) can functionally replace the N terminus of SP-A for lipid aggregation and tubular myelin formation, but not for surface tension lowering properties of SP-A, and 2) is not sufficient to reverse the structural and metabolic pulmonary defects in the SP-D(-/-) mouse.     

Distinct effects of surfactant protein A or D deficiency during bacterial infection on the lung

Mice lacking surfactant protein (SP)-A (SP-A-/-) or SP-D (SP-D-/-) and wild-type mice were infected with group B streptococcus or Haemophilus influenzae by intratracheal instillation. Although decreased killing of group B streptococcus and H. influenzae was observed in SP-A-/- mice but not in SP-D-/- mice, deficiency of either SP-A or SP-D was associated with increased inflammation and inflammatory cell recruitment in the lung after infection. Deficient uptake of bacteria by alveolar macrophages was observed in both SP-A- and SP-D-deficient mice. Isolated alveolar macrophages from SP-A-/- mice generated significantly less, whereas those from SP-D-/- mice generated significantly greater superoxide and hydrogen peroxide compared with wild-type alveolar macrophages. In SP-D-/- mice, bacterial killing was associated with increased lung inflammation, increased oxidant production, and decreased macrophage phagocytosis. In contrast, in the absence of SP-A, bacterial killing was decreased and associated with increased lung inflammation, decreased oxidant production, and decreased macrophage phagocytosis. Increased oxidant production likely contributes to effective bacterial killing in the lungs of SP-D-/- mice. The collectins, SP-A and SP-D, play distinct roles during bacterial infection of the lung.     

Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages

Surfactant protein-A (SP-A) plays multiple roles in pulmonary host defense, including stimulating bacterial phagocytosis by innate immune cells. Previously, SP-A was shown to interact with complement protein C1q. Our goal was to further characterize this interaction and elucidate its functional consequences. Radiolabeled SP-A bound solid-phase C1q but not other complement proteins tested. The lectin activity of SP-A was not required for binding to C1q. Because C1q is involved in bacterial clearance but alone does not efficiently enhance the phagocytosis of most bacteria, we hypothesize that SP-A enhances phagocytosis of C1q-coated antigens. SP-A enhanced by sixfold the percentage of rat alveolar macrophages in suspension that phagocytosed C1q-coated fluorescent beads. Furthermore, uptake of C1q-coated beads was enhanced when either beads or alveolar macrophages were preincubated with SP-A. In contrast, SP-A had no significant effect on the uptake of C1q-coated beads by alveolar macrophages adhered to plastic slides. We conclude that SP-A may serve a protective role in the lung by interacting with C1q to enhance the clearance of foreign particles.     

Characterization of pulmonary surfactant protein D: its copurification with lipids

Surfactant protein D (SP-D) is a collagenous surfactant associated protein synthesized by alveolar type II cells. SP-D was purified from the supernatant of rat bronchoalveolar lavage fluids obtained by centrifugation at 33,000 x gav for 16 h. The contents of SP-D and SP-A in fractions obtained by the centrifugation of rat bronchoalveolar lavage were determined by enzyme-linked immunoassay. The total content of SP-D was approximately 12% of that of SP-A in these lavage fluids. 99.1% of SP-A was present in the 33,000g pellet, whereas 71.1% of SP-D was in the 33,000g supernatant. Analysis by high performance liquid chromatography reveals that lipids are copurified with isolated SP-D. Phosphatidylcholine accounted for 84.8% of the phospholipids copurified with SP-D. Unlike SP-A, SP-D in the purified and delipidated form failed to compete with 125I-labeled SP-A for phosphatidylcholine binding, and to aggregate phospholipid liposomes. The present study demonstrates that lipids are copurified with SP-D, that SP-D and SP-A distribute differently in rat bronchoalveolar lavage fluids, and that SP-D in the purified and delipidated form does not exhibit interaction with lipids in the same fashion as SP-A.     

L-Arg对LPS诱导的急性肺损伤大鼠肺表面活性物质和肺泡巨噬细胞功能的影响

目的:探讨L-精氨酸(L-Arg)对脂多糖(LPS)诱导的急性肺损伤大鼠肺表面活性物质和肺泡巨噬细胞功能的影响。方法:舌下静脉注射脂多糖(LPS)复制肺损伤模型。健康雄性SD大鼠48只,随机分为对照组、模型组(LPS组)和L-Arg治疗组(L-Arg组)(n=16)。分别于给予LPS 3 h或6 h后给予生理盐水(对照组及LPS组,ip)和L-Arg(500 mg/kg ip)(L-Arg治疗组),治疗3 h。原位杂交法(ISH)检测肺组织中肺表面活性蛋白A(SP-A)mRNA的表达;测定肺泡灌洗液(BALF)中的总蛋白(TP)。体外分离培养大鼠肺泡巨噬细胞,以LPS(终浓度10 mg/L)处理巨噬细胞,观察L-Arg对肺泡巨噬细胞的影响。结果:与对照组比较,大鼠肺损伤后SP-A mRNA表达减弱,BALF中TP增多(P<0.01)。肺损伤3 h用L-Arg治疗3 h后,SP-A mRNA阳性细胞表达明显增强,BALF中TP较LPS组相同时间点明显降低(P<0.05,P<0.01),肺损伤减轻。体外实验中,与正常对照组相比,LPS组细胞培养上清中乳酸脱氢酶(LDH)、一氧化氮(NO)、肿瘤坏死因子-α(TNFα-)和白细胞介素-6(IL-6)浓度明显增高(P<0.01);L-Arg明显减少LPS所致的LDH的释放,降低TNFα-和IL-6浓度。结论:L-Arg可减轻内毒素性肺损伤,此机制可能与增强SP-AmRNA表达有关;LPS可刺激巨噬细胞分泌促炎因子和NO,L-Arg可抑制LPS对巨噬细胞的作用。     

Surfactant proteins A and D enhance the phagocytosis of Chlamydia into THP-1 cells

Chlamydiae are intracellular bacterial pathogens that infect mucosal surfaces, i.e., the epithelium of the lung, genital tract, and conjunctiva of the eye, as well as alveolar macrophages. In the present study, we show that pulmonary surfactant protein A (SP-A) and surfactant protein D (SP-D), lung collectins involved in innate host defense, enhance the phagocytosis of Chlamydia pneumoniae and Chlamydia trachomatis by THP-1 cells, a human monocyte/macrophage cell line. We also show that SP-A is able to aggregate both C. trachomatis and C. pneumoniae but that SP-D only aggregates C. pneumoniae. In addition, we found that after phagocytosis in the presence of SP-A, the number of viable C. trachomatis pathogens in the THP-1 cells 48 h later was increased approximately 3.5-fold. These findings suggest that SP-A and SP-D interact with chlamydial pathogens and enhance their phagocytosis into macrophages. In addition, the chlamydial pathogens internalized in the presence of collectins are able to grow and replicate in the THP-1 cells after phagocytosis.     

Macrophage and type II cell catabolism of SP-A and saturated phosphatidylcholine in mouse lungs

Type II cells and macrophages are the major cells involved in the alveolar clearance and catabolism of surfactant. We measured type II cell and macrophage contributions to the catabolism of saturated phosphatidylcholine and surfactant protein A (SP-A) in mice. We used intratracheally administered SP-A labeled with residualizing (125)I-dilactitol-tyramine, radiolabeled dipalmitoylphosphatidylcholine ([(3)H]DPPC), and its degradation-resistant analog [(14)C]DPPC-ether. At 15 min and 7, 19, 29, and 48 h after intratracheal injection, the mice were killed; alveolar lavage was then performed to recover macrophages and surfactant. Type II cells and macrophages not recovered by the lavage were subsequently isolated by enzymatic digestion of the lung. Radioactivity was measured in total lung, lavage fluid macrophages, alveolar washes, type II cells, and lung digest macrophages. Approximately equal amounts of (125)I-dilactitol-tyramine-SP-A and [(14)C]DPPC-ether associated with the macrophages (lavage fluid plus lung digest) and type II cells when corrected for the efficiency of type II cell isolation. Eighty percent of the macrophage-associated radiolabel was recovered from lung digest macrophages. We conclude that macrophages and type II cells contribute equally to saturated phosphatidylcholine and SP-A catabolism in mice.     

Pulmonary surfactant protein A up-regulates activity of the mannose receptor,a pattern recognition receptor expressed on human macrophages

Inhaled particulates and microbes are continually cleared by a complex array of lung innate immune determinants, including alveolar macrophages (AMs). AMs are unique cells with an enhanced capacity for phagocytosis that is due, in part, to increased activity of the macrophage mannose receptor (MR), a pattern recognition receptor for various microorganisms. The local factors that "shape" AM function are not well understood. Surfactant protein A (SP-A), a major component of lung surfactant, participates in the innate immune response and can enhance phagocytosis. Here we show that SP-A selectively enhances MR expression on human monocyte-derived macrophages, a process involving both the attached sugars and collagen-like domain of SP-A. The newly expressed MR is functional. Monocyte-derived macrophages on an SP-A substrate demonstrated enhanced pinocytosis of mannose BSA and phagocytosis of Mycobacterium tuberculosis lipoarabinomannan-coated microspheres. The newly expressed MR likely came from intracellular pools because: 1) up-regulation of the MR by SP-A occurred by 1 h, 2) new protein synthesis was not necessary for MR up-regulation, and 3) pinocytosis of mannose BSA via MR recycling was increased. AMs from SP-A(-/-) mice have reduced MR expression relative to SP-A(+/+). SP-A up-regulation of MR activity provides a mechanism for enhanced phagocytosis of microbes by AMs, thereby enhancing lung host defense against extracellular pathogens or, paradoxically, enhancing the potential for intracellular pathogens to enter their intracellular niche. SP-A contributes to the alternative activation state of the AM in the lung.     

Surfactant protein D increases phagocytosis and aggregation of pollen-allergen starch granules

Recent studies have shown that surfactant components, in particular the collectins surfactant protein (SP)-A and -D, modulate the phagocytosis of various pathogens by alveolar macrophages. This interaction might be important not only for the elimination of pathogens but also for the elimination of inhaled allergens and might explain anti-inflammatory effects of SP-A and SP-D in allergic airway inflammation. We investigated the effect of surfactant components on the phagocytosis of allergen-containing pollen starch granules (PSG) by alveolar macrophages. PSG were isolated from Dactylis glomerata or Phleum pratense, two common grass pollen allergens, and incubated with either rat or human alveolar macrophages in the presence of recombinant human SP-A, SP-A purified from patients suffering from alveolar proteinosis, a recombinant fragment of human SP-D, dodecameric recombinant rat SP-D, or the commercially available surfactant preparations Curosurf and Alveofact. Dodecameric rat recombinant SP-D enhanced binding and phagocytosis of the PSG by alveolar macrophages, whereas the recombinant fragment of human SP-D, SP-A, or the surfactant lipid preparations had no effect. In addition, recombinant rat SP-D bound to the surface of the PSG and induced aggregation. Binding, aggregation, and enhancement of phagocytosis by recombinant rat SP-D was completely blocked by EDTA and inhibited by d-maltose and to a lesser extent by d-galactose, indicating the involvement of the carbohydrate recognition domain of SP-D in these functions. The modulation of allergen phagocytosis by SP-D might play an important role in allergen clearance from the lung and thereby modulate the allergic inflammation of asthma.     

Simultaneous absence of surfactant proteins A and D increases lung inflammation and injury after allogeneic HSCT in mice

The relative contributions of the hydrophilic surfactant proteins (SP)-A and -D to early inflammatory responses associated with lung dysfunction after experimental allogeneic hematopoietic stem cell transplantation (HSCT) were investigated. We hypothesized that the absence of SP-A and SP-D would exaggerate allogeneic T cell-dependent inflammation and exacerbate lung injury. Wild-type, SP-D-deficient (SP-D(-/-)), and SP-A and -D double knockout (SP-A/D(-/-)) C57BL/6 mice were lethally conditioned with cyclophosphamide and total body irradiation and given allogeneic bone marrow plus donor spleen T cells, simulating clinical HSCT regimens. On day 7, after HSCT, permeability edema progressively increased in SP-D(-/-) and SP-A/D(-/-) mice. Allogeneic T cell-dependent inflammatory responses were also increased in SP-D(-/-) and SP-A/D(-/-) mice, but the altered mediators of inflammation were not identical. Compared with wild-type, bronchoalveolar lavage fluid (BALF) levels of nitrite plus nitrate, GM-CSF, and MCP-1, but not TNF-alpha and IFN-gamma, were higher in SP-D-deficient mice before and after HSCT. In SP-A/D(-/-) mice, day 7 post-HSCT BALF levels of TNF-alpha and IFN-gamma, in addition to nitrite plus nitrate and MCP-1, were higher compared with mice lacking SP-D alone. After HSCT, both SP-A and SP-D exhibited anti-inflammatory lung-protective functions that were not completely redundant in vivo.     

Interaction of surfactant protein A with peroxiredoxin 6 regulates phospholipase A2 activity

Peroxiredoxin 6 (Prdx6) is a "moonlighting" protein with both GSH peroxidase and phospholipase A(2) (PLA(2)) activities. This protein is responsible for degradation of internalized dipalmitoylphosphatidylcholine, the major phospholipid component of lung surfactant. The PLA(2) activity is inhibited by surfactant protein A (SP-A). We postulate that SP-A regulates the PLA(2) activity of Prdx6 through direct protein-protein interaction. Recombinant human Prdx6 and SP-A isolated from human alveolar proteinosis fluid were studied. Measurement of kinetic constants at pH 4.0 (maximal PLA(2) activity) showed K(m)0.35 mm and V(max) 138 nmol/min/mg of protein. SP-A inhibited PLA(2) activity non-competitively with K(i) 10 mug/ml and was Ca(2+) -independent. Activity at pH 7.4 was approximately 50% less, and inhibition by SP-A was partially dependent on Ca(2+). Interaction of SP-A and Prdx6 at pH 7.4 was shown by Prdx6-mediated inhibition of SP-A binding to agarose beads, a pull-down assay using His-tagged Prdx6 and Ni(2) -chelating beads, co-immunoprecipitation from lung epithelial cells and from a binary mixture of the two proteins, binding after treatment with a trifunctional cross-linker, and size-exclusion chromatography. Analysis by static light scattering and surface plasmon resonance showed calcium-independent SP-A binding to Prdx6 at pH 4.0 and partial Ca(2+) dependence of binding at pH 7.4. These results indicate a direct interaction between SP-A and Prdx6, which provides a mechanism for regulation of the PLA(2) activity of Prdx6 by SP-A.     

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.     

Surfactant protein A is localized at the corners of the pulmonary tubular myelin lattice

Immunogold labeling on sections of a freeze-substituted tubular myelin-enriched fraction isolated from a bronchoalveolar lavage of rat lung showed that surfactant protein A (SP-A) occurs predominantly at the corners of the tubular myelin lattice. Seventy-nine percent of the gold particles were located within 20 nm from a corner. Extracellular SP-A was detected only in the tubular myelin lattice and not in vesicles or secreted lamellar bodies. Ultra-thin cryosections of rat lung fixed in vivo showed that intracellular SP-A was distributed homogeneously over the stacked membranes of lamellar bodies in alveolar Type II cells. The presence of SP-A at the corners of the tubular myelin lattice suggests an important role of this protein in the formation and/or maintenance of this highly ordered lattice.     

A phagocytosis assay for oxidized low-density lipoprotein versus immunoglobulin G-coated microbeads in human U937 macrophages

The human monocyte cell line U937 was differentiated into an adherent macrophage phenotype using phorbol 12-myristate 13-acetate (PMA) to assay the phagocytosis of oxidized low-density lipoprotein (oxLDL) that may play a role in atherosclerosis. Microbeads were coated with the inflammatory ligand oxLDL to create a novel phagocytosis assay that models the binding of macrophages to oxLDL in the solid phase such as found in the fatty streaks of the arteries. The oxLDL was prepared with LDL from human ethylenediaminetetraacetic acid (EDTA) plasma oxidized with an excess (5 mM) of the strong oxidizing agent CuSO₄ and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis with Western blot. The binding of the oxLDL to the beads was confirmed by DilC18–oxLDL staining and confocal microscopy in addition to trypsin digestion of the microbeads for liquid chromatography, electrospray ionization, and tandem mass spectrometry. Phagocytosis of the oxLDL versus human bulk immunoglobulin G1 (IgG1)-coated microbeads was assayed over time, in the presence and absence of serum factors, by pulse chase and with enzyme inhibitor treatments. The ligand beads were then stained with specific antibodies to oxLDL versus human IgG to differentially stain external versus engulfed ligand microbeads. The phagocytosis of oxLDL and IgG ligand microbeads was abolished by the actin polymerization inhibitors cytochalasin D and latrunculin. Pharmacological inhibitors of the receptor enzymes JAK, SRC, and PLC prevented both IgG and oxLDL receptor function. In contrast, the function of the oxLDL phagocytic receptor complex was more sensitive to inhibition of PTK2, PKC, and SYK activity.     

Surfactant protein A enhances mycobacterial killing by rat macrophages through a nitric oxide-dependent pathway

Surfactant-associated protein A (SP-A) is involved in surfactant homeostasis and host defense in the lung. We have previously demonstrated that SP-A specifically binds to and enhances the ingestion of bacillus Calmette-Guerin (BCG) organisms by macrophages. In the current study, we investigated the effect of SP-A on the generation of inflammatory mediators induced by BCG and the subsequent fate of ingested BCG organisms. Rat macrophages were incubated with BCG in the presence and absence of SP-A. Noningested BCG organisms were removed, and the release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide were measured at varying times. TNF-alpha and nitric oxide production induced by BCG were enhanced by SP-A. In addition, SP-A enhanced the BCG-induced increase in the level of inducible nitric oxide synthase protein. Addition of antibodies directed against SPR210, a specific macrophage SP-A receptor, inhibited the SP-A-enhanced mediator production. BCG in the absence of SP-A showed increased growth over a 5-day period, whereas inclusion of SP-A dramatically inhibited BCG growth. Inhibition of nitric oxide production blocked BCG killing in the presence and absence of SP-A. These results demonstrate that ingestion of SP-A-BCG complexes by rat macrophages leads to production of inflammatory mediators and increased mycobacterial killing.     

Quantification of surfactant pool sizes in rabbit lung during perinatal development

Methods are presented for the quantitative isolation of surfactants from fetal and newborn rabbit alveolar lavage returns and post-lavaged lung tissue homogenates. The phospholipid content of both fractions progressively increased between 27 days gestation and term (31 days). The tissue-stored fraction increased approximately 16-fold (from 0.48 +/- 0.13 to 7.83 +/- 0.86 mg/g dry lung) and the alveolar fraction more than 30-fold (from 0.08 +/- 0.02 to 2.69 +/- 0.52 mg/g dry lung). Developmental changes in phospholipid composition were also observed. Tissue-stored surfactant was prepared using differential and density gradient centrifugation. Alveolar surfactant was isolated during fetal development as a high-speed pellet following a one-step differential centrifugation. There was little change in the phospholipid content of fetal alveolar lavage supernatant (range 0.12 +/- 0.04 to 0.28 +/- 0.09 mg/g dry lung). By the first postnatal day the phospholipid content of both lavage fractions significantly increased (pellet, 7.51 +/- 1.79; supernatant, 4.01 +/- 1.36 mg/g dry lung) and both were identified as surfactant. This increase in alveolar surfactant was accompanied by an approximately twofold decrease (to 3.81 +/- 1.1 mg/g dry lung) in the tissue-stored fraction. These data provide a quantitative profile of surfactant accumulation and secretion in developing rabbit lung.