Migratory behaviors of alveolar macrophages during the alveolar clearance of light to heavy burdens of particles

We investigated the unstimulated and stimulated migratory activities of lavaged alveolar macrophages (AMs) in vitro over the course of alveolar clearance of three different mass lung burdens of microspheres. Our intent was to uncover potentially important relationships between the migratory behaviors of the AM and the retention kinetics of particles. Groups of adult, male Fischer-344 rats were intratracheally instilled with approximately 86 micrograms (low burden, LB), approximately 1 mg (medium burden, MB), or approximately 3.7 mg (high burden, HB) of polystyrene microspheres (2.13 microns diameter), or with carrier vehicle (phosphate buffered saline, PBS) alone. The lung retention kinetics of the particles were determined over an approximately 170 day period. On days 14, approximately 57, and approximately 85, lavaged AMs were assessed for their abilities to migrate through 5-microns pore membranes in response to inactivated rat serum (unstimulated condition) and yeast-activated rat serum (stimulated condition). The retention characteristics of the three burdens could be satisfactorily described by two-component, negative exponential equations. The kinetics of retention of the LB and MB were similar, although some evidence indicated the MB slightly retarded the lung clearance process. Deposition of the HB resulted in more marked prolongations of both the early, more rapid, and the slower, longer term components of alveolar clearance. The unstimulated migration indices of AMs from the particle-instilled lungs were generally not significantly different from those of AMs from PBS-instilled lungs except for a significant increase in the migration indices of LB AMs at the last assay time. The stimulated migration indices of MB and HB AMs were significantly decreased on assay days 14 and approximately 57. On day approximately 85, however, the migration indices of LB, MB, and HB AMs were all increased above the PBS AMs. Comparisons of the frequency distributions of particles in the unstimulated and stimulated AM that migrated to those in corresponding parent AM populations consistently indicated a preferential migration of particle-free AMs and of AMs with lesser loads of microspheres. The overall results of this study suggest that the unstimulated and stimulated migratory activities of particle-laden AMs are depressed in vitro. Our results also suggest that the migratory activities of generally particle-free AMs may be enhanced over a prolonged period of time following the deposition of particles in the lung.     

Human alveolar long-term clearance of ferromagnetic iron oxide microparticles in healthy and diseased subjects

Monodisperse ferrimagnetic microparticles (Fe3O4) with 1.3 microm geometric diameter were inhaled to study alveolar long-term clearance in healthy and diseased human subjects. Nineteen younger (age 20 to 39 years) and 20 older (age 40 to 65 years) healthy volunteers participated in the study as well as 15 patients with sarcoidosis (SAR), 12 patients with idiopathic pulmonary fibrosis (IPF), and 15 patients with chronic obstructive bronchitis (COB). In each group the subjects were divided into never smokers (NS) and active smokers (S). Clearance was measured by magnetopneumography (MPG) for 300 days after inhalation. In COB, 50% of the deposited particles were removed from the lungs after 2 days, indicating high bronchial deposits due to bronchial obstructions. In healthy NS, only 10% of the particles were removed after 2 days and cigarette smoking enhanced the fraction of fast-cleared particles. In subjects who smoked, slow clearance was significantly impaired (P < . 02). Clearance half-lives (in days) for younger, healthy, NS were 124 +/- 66 (mean +/- SD) compared to 220 +/- 74 for S. Similarly for older subjects, the timeswere 162 +/- 120 for NS and 459 +/- 334 for S. The impairment of alveolar clearance due to cigarette smoking increases by 5.7 +/- 1.3 days/pack-year (P < .01). Alveolar clearance was impaired in SAR and in IPF; half-lives were 275 +/- 109 days (P < .05) and 756 +/- 345 days (P < .02), respectively, compared to healthy NS. Most COB patients were ex-smokers, their long-term clearance was 240 +/- 74 days, which is more than healthy NS (P < .01), but less than healthy S and might indicate a recovery of alveolar clearance. In view of studies using totally inert particles like Teflon, we conclude that the lung clearance measured with iron oxide tracer particles primarily reflects clearance by intraphagosomal particle dissolution within alveolar macrophages, which is impaired by cigarette smoke consumption and in patients.     

Enhanced alveolar clearance with chest percussion therapy and positional changes during whole-lung lavage for alveolar proteinosis

Pulmonary alveolar proteinosis has traditionally been treated with whole-lung lavage (WLL). The literature describes a variety of techniques used in performing the WLL, including mechanical vs manual chest percussion, use of prone positioning, and variances in lavage volume. We have quantified and compared the effective alveolar clearance for each component of the lavage by measuring the dry weight of material in the lavage effluent. We measured this in five patients who underwent six consecutive WLLs at the University of Pittsburgh Medical Center. We performed the lavage in the following three stages: stage I, passive drainage; stage II, assisted clearance; and stage III, positional clearance. Aliquots of lavage effluent were centrifuged to determine the dry weight of material present in sequentially recorded bottles within each stage. At the initiation of each augmentation, there was a statistically significant improvement in the clearance of material (stage II, p = 0.009; stage III, p = 0.012). Furthermore, we show that lipoproteinaceous material is present in the lavage effluent in all stages of latter portions of the lavage. The effective removal of material would be expected to have an impact on the physiologic and clinical response to WLL. This finding emphasizes the importance of performing an adequate and standardized lavage.     

Effect of aging on nitric oxide production by rat alveolar macrophages

Nitric oxide (NO) plays an important role in alveolar macrophages (AM)-mediated defense against infection. The elderly become highly susceptible to respiratory tract infection. Inhibition of NO production significantly suppresses defense against infections. Therefore, it is necessary to elucidate the effect of senescence on NO production of AM. The alveolar microenvironment and lymphocytes affect NO production by AM. We examined whether changes in the alveolar microenvironment, lymphocytes, or AM brought about by aging affect NO production by AM. Bronchoalveolar lavage fluid was used as a substitute for the alveolar microenvironment. The results showed that NO production by AM activated by lymph node cells in bronchoalveolar lavage fluid from old rats in response to concanavalin A decreased compared with that of young rats. AM from aged rats produced less NO than AM from young rats. Bronchoalveolar lavage fluid and lymph node cells from aged rats had no effect on the amount of NO produced by AM. Therefore, age-associated decrease in the functional capacity of AM plays a central role in the decrease of NO production.     

Parallel induction of nitric oxide and tetrahydrobiopterin synthesis in alveolar macrophages.

BACKGROUND: Nitric oxide (NO) and an essential cofactor for both constitutive and inducible NO synthase (NOS) activity, tetrahydrobiopterin (6R-L-erythro-1',2'-dihydroxypropyl-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine; BH4), are thought to be important modulators of function in normal and inflamed airways. However, the exact pathologic roles of NO and BH4 remain obscure. Even less is known about the effects of cytokines on alveolar macrophages. OBJECTIVE: This study was designed to determine whether NO and BH4 are induced by cytokines in mouse alveolar macrophages and to investigate whether NO synthesis is affected by changes in intracellular BH4 levels in alveolar macrophages. METHODS: We compared the induction by lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-2 (IL-2) of NO production and BH4 synthesis in alveolar macrophages. To determine whether NO synthesis is affected by changes in intracellular BH4 levels in alveolar macrophages, we used inhibitors of BH4 biosynthesis. RESULTS: Activation of alveolar macrophages induced parallel increases in NO and intracellular BH4 levels, although induction of the latter appears to be somewhat more sensitive than that of the latter to diverse cytokines. Inducible NO production in alveolar macrophages was blocked by inhibitors of BH4 biosynthesis. IL-2, an important component of the immunomodulatory system, was only a weak activator of alveolar macrophages by itself but potently synergized with IFN-gamma to stimulate the production of both NO and BH4. CONCLUSION: Our results suggest that BH4 synthesis in alveolar macrophages is a potential target for therapeutic intervention in airway inflammatory diseases, such as asthma, cystic fibrosis, and acute bronchial infections whose pathology may be mediated by overproduction of NO.     

肺泡上皮在肺液体平衡中的作用

肺泡上皮的一个重要作用是组成肺泡毛细血管屏障，分泌表面活性物质降低肺表面张力，清除肺水肿。肺泡液体的转运是一个主动耗能过程，它是由于钠离子的主动重吸收产生了一个浓度梯度从而引起水的被动吸收。主动钠转运通过腔膜侧的钠通道和氯通道及基底膜侧的Na^＋-K＋-ATP酶调节。研究肺泡上皮细胞液体转运对肺水肿的治疗有重要临床意义。     

Macrolide antibiotics inhibit nitric oxide generation by rat pulmonary alveolar macrophages.

There is evidence that macrolide antibiotics are effective in the treatment of chronic airway inflammatory diseases, probably through actions other than their antibacterial properties. In order to determine whether macrolides affect the nitric oxide-generating system in the respiratory tract, rat pulmonary alveolar macrophages (PAMs) were studied in vitro. The release of NO was assessed by direct measurement with a specific amperometric sensor for this molecule, and the expression of type II NO synthase (NOS) messenger ribonucleic acid (mRNA) was determined by Northern blotting. Incubation of PAMs with lipopolysaccharide from Escherichia coli and recombinant human interferon-gamma caused release of NO, which was accompanied by induction of type II NOS mRNA. The release of NO was reduced by coincubation of cells with the macrolides erythromycin, clarithromycin and josamycin in a concentration-dependent manner, the maximal inhibition being 73+/-10, 81+/-6 and 84+/-9%, respectively, but was not altered by amoxycillin or cefaclor. These macrolides likewise inhibited the induction of type II NOS mRNA, whereas no inhibitory effects were observed with amoxycillin or cefaclor. These results suggest that macrolide antibiotics specifically inhibit type II NO synthase gene expression and consequently reduce NO production by rat pulmonary alveolar macrophages, which might result in attenuation of airway inflammation.     

Proliferation of pulmonary alveolar macrophages during postnatal development of rabbit lungs

The purpose of this study was to determine if proliferation of pulmonary alveolar macrophages (PAM) played a significant role in establishing the PAM population of the lung during postnatal development. New Zealand albino rabbits were killed at 0.5, 1 through 5, 7, and 14 days and 4 months after birth and their lungs were lavaged. Cell yield in the lavage was determined by hemocytometer counts, and the percentage of PAM in mitosis (mitotic index) was determined from cytocentrifuge preparations. The total number of PAM increased from 1.5 X 10(6) at 1 day to 8.38 X 10(6) at 14 days after birth. The mitotic index (MI) was 0.6% at 0.5 days after birth, increased to 1.6% at 1 day, and remained elevated through 5 days. By 14 days, the MI declined to 0.2%. The cell cycle time (Ct) of the PAM population was calculated from the MI and ranged from 1.8 to 2.4 days during Days 1 through 5 of life. Direct measurements of the doubling time (Dt) of PAM in the lavage revealed that the PAM population doubled twice over this same time period. Because Ct was equal to Dt during Days 1 through 5, we conclude that proliferation of PAM was the primary mechanism by which the PAM population increased during the immediate postnatal development of the rabbit lung. No evidence was obtained indicating that migratory monocytes or interstitial macrophages were involved with this process of population expansion. This study adds to the growing literature demonstrating that the intraalveolar proliferation of "free" PAM is the major local source of PAM in the lung.     

Lysosomal enzyme activities in pulmonary macrophages from rabbits breathing iron oxide

The effects of an in vivo exposure to a nontoxic particle, iron oxide, on lysosomal hydrolases in pulmonary macrophages were examined. Rabbits breathed a submicron-sized aerosol of iron oxide for 3 h (mass concentration, 186 to 222 mg/m3). Macrophages were recovered by lung lavage 0, 12, 18, and 24 h later. The number of cells lavaged after iron oxide was significantly greater than the control number, whereas the amount of protein recovered per 10(8) cells decreased, suggesting the influx of smaller cells. Macrophages from animals exposed to iron oxide reacted histochemically for acid phosphatase showed increased stain intensity and redistribution of enzyme within the cytoplasm. Considerable cell-to-cell variability in enzyme activity was evident in these macrophages. Homogenates of cells exposed to iron oxide assayed for 6 lysosomal acid hydrolases showed little change in specific activity or in total enzyme per 10(8) cells when compared to homogenates of cells from control animals. Sucrose density gradient centrifugation demonstrated that exposure to iron oxide caused an increase in lysosome heterogeneity over that seen in control preparations and the appearance of a population of lysosomes of increased density. An aerosol of iron oxide did not greatly alter the average concentrations of lysosomal enzymes in pulmonary macrophages, but did stimulate recruitment of new cells and thus increases the total amount of some enzymes in the lung.     

Early changes in alveolar fluid clearance by nitric oxide after endotoxin instillation in rats

Alveolar fluid clearance may be inhibited and/or stimulated under pathologic conditions. We examined the early change of alveolar fluid clearance after endotoxin instillation in adult rats. We employed electron paramagnetic resonance nitric oxide (NO) trapping technique with iron complex with N,N-diethyldithiocarbamate as an NO trapping agent. We found that lung NO signals reached the highest magnitude by 6 hours after endotoxin instillation. NO production was accompanied by increases in lung cyclic guanosine monophosphate levels. Alveolar fluid clearance decreased significantly 6 hours after the administration of the endotoxin and increased further at 24 hours. These changes were shown to be related to the function of amiloride-sensitive sodium ion channels. Treatment with gadolinium chloride and aminoguanidine significantly decreased lung NO and cyclic guanosine monophosphate levels and completely ameliorated the decrease in alveolar fluid clearance. In addition, the increase in alveolar fluid clearance at 24 hours returned to normal levels after treatment with gadolinium chloride and aminoguanidine. We found immunoreactive inducible nitric oxide synthase to be abundantly expressed in the cytoplasm of alveolar macrophages. Our results suggest that alveolar endotoxin inhibits alveolar fluid clearance at 6 hours by NO. NO is produced via inducible NO synthase in endotoxin-stimulated alveolar macrophages and was also shown to increase alveolar fluid clearance at 24 hours.     

脂多糖对大鼠肺泡巨噬细胞分泌一氧化氮和氧化应激的影响

目的探讨细菌内毒素脂多糖（LPS）对SD大鼠肺泡巨噬细胞产生一氧化氮（NO）和氧化应激的影响。方法采用支气管肺泡灌洗和细胞差速贴壁的方法分离大鼠肺泡巨噬细胞（AM）,分别测定AM培养上清液NO含量、一氧化氮合酶（NOS）活性、丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性。结果在5,10,20,50mg/LLPS分别干预下,大鼠AM培养上清液NO含量、NOS活性和MDA含量均显著升高,SOD活性显著降低,并且具有浓度依赖性。结论LPS促进大鼠AM分泌NO,并诱导AM脂质过氧化损伤,这可能是内毒素诱发肺部炎症反应不易控制和急性肺损伤的机制之一。     

Adenosine regulation of alveolar fluid clearance

Adenosine is a purine nucleoside that regulates cell function through G protein-coupled receptors that activate or inhibit adenylyl cyclase. Based on the understanding that cAMP regulates alveolar epithelial active Na(+) transport, we hypothesized that adenosine and its receptors have the potential to regulate alveolar ion transport and airspace fluid content. Herein, we report that type 1 (A(1)R), 2a (A(2a)R), 2b (A(2b)R), and 3 (A(3)R) adenosine receptors are present in rat and mouse lungs and alveolar type 1 and 2 epithelial cells (AT1 and AT2). Rat AT2 cells generated and produced cAMP in response to adenosine, and micromolar concentrations of adenosine were measured in bronchoalveolar lavage fluid from mice. Ussing chamber studies of rat AT2 cells indicated that adenosine affects ion transport through engagement of A(1)R, A(2a)R, and/or A(3)R through a mechanism that increases CFTR and amiloride-sensitive channel function. Intratracheal instillation of low concentrations of adenosine (< or =10(-8)M) or either A(2a)R- or A(3)R-specific agonists increased alveolar fluid clearance (AFC), whereas physiologic concentrations of adenosine (> or =10(-6)M) reduced AFC in mice and rats via an A(1)R-dependent pathway. Instillation of a CFTR inhibitor (CFTR(inh-172)) attenuated adenosine-mediated down-regulation of AFC, suggesting that adenosine causes Cl(-) efflux by means of CFTR. These studies report a role for adenosine in regulation of alveolar ion transport and fluid clearance. These findings suggest that physiologic concentrations of adenosine allow the alveolar epithelium to counterbalance active Na(+) absorption with Cl(-) efflux through engagement of the A(1)R and raise the possibility that adenosine receptor ligands can be used to treat pulmonary edema.     

Hydrolytic enzymes of alveolar macrophages

Hydrolytic enzymes are major constituents of alveolar macrophages, which in recent years have been shown to be involved in many aspects of the inflammatory response in addition to their better-known role in bactericidal processes. This review summarizes the general properties, physiologic function, cellular physiology, and clinical associations of four important hydrolytic enzymes of alveolar macrophages--lysozyme, elastase, plasminogen activator, and collagenase--with particular attention to the relationship of these enzymes to the pathophysiology of lung disease. The information reviewed shows that much is known about the biochemistry of these enzymes, that each is produced in greater quantity when alveolar macrophages are stimulated, that each has a distinctive physiologic role in the inflammatory process, and that they function as part of the overall pulmonary antibacterial defense system. Studies of the pathophysiologic effects consequent to the elaboration of excess quantities of these enzymes by stimulated macrophages show that some hydrolytic enzymes injure the lung by attacking normal as well as inflammatory tissue sites that are susceptible to degradation. Such damage is normally limited by enzymatic inhibitors, like alpha-antitrypsin, but the inactivating capacity of the inhibitors can be overwhelmed and in these instances excess enzyme contributes to the development of emphysema. This newer understanding of the pathophysiologic role of hydrolytic enzymes may lead to therapeutically beneficial methods for modulating the pulmonary inflammatory response.     

Volumetric loading of alveolar macrophages (AM): a possible basis for diminished AM-mediated particle clearance.

Using intratracheal instillation of radioactively labeled plastic microspheres of 3.3 and 10.3 microns diameter at two dose levels, this 7-month study in male Fischer 344 rats was designed to test a volumetric particulate burden hypothesis that has been proposed as a mechanistic basis for the condition of dust overloading of the lungs with highly insoluble particles of very low toxicity and to explain the prolongation of pulmonary particle retention. The study utilized airway and deep lung lavage techniques, scanning electron and optical microscopy of lavaged cells and lungs of sacrificed animals, particle distribution in alveolar macrophages (AM), fecal recovery of radioactive particles, and lung retention measurements by external counting. Microscopic assessments revealed that essentially all of the 3.3- and 10.3-microns-diameter particles were phagocytized by AM within 24 h postinstillation. One phagocytized 10.3-microns particle is capable of producing the hypothesized 600-microns 3/AM overload criterion for virtual AM immobilization. Neither the number nor the volume of 3.3-microns-diameter particles instilled was large enough to produce volumetric overloading assuming uniform distribution of the particles in the lung. In contrast to the 3.3-microns particles, the 10.3-microns particles were apparently sequestered to a greater extent and capable of greatly prolonging AM-mediated clearance of particles from the pulmonary region. The measured pulmonary retention half-times for the small and large particles were 86-109 days and 870-1020 days, respectively. Fecal recovery data closely complemented pulmonary clearance data for both particle sizes. The two-particle approach was found supportive of the volumetric overload hypothesis.     

Pulmonary intravascular macrophages metabolize arachidonic acid in vitro. Comparison with alveolar macrophages

Pulmonary intravascular macrophages are a recently identified component of the pulmonary mononuclear phagocyte system. It has been shown that alveolar macrophages are capable of metabolizing arachidonic acid (AA) to its biologically active inflammatory metabolites via the lipoxygenase and cyclooxygenase pathways. In this study, we have compared the ability of swine intravascular macrophages and alveolar macrophages to metabolize AA in vitro. Alveolar macrophages attached to a plastic substrate produced at least five identified AA metabolites including prostaglandin (PG)F2 alpha, hydroxyheptadecatrienoic acid (HHT), 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, and 15-HETE. In contrast, adherent intravascular macrophages produced eight identified metabolites including thromboxane (TX)B2, PGF2 alpha, PGD2, PGE2, HHT, 5-HETE, 12-HETE, and 15-HETE. The major lipoxygenase metabolite produced by both macrophage types was 5-HETE. The major cyclooxygenase metabolite produced by alveolar macrophages was PGF2 alpha, whereas the major metabolite produced by intravascular macrophages was HHT. Both macrophage populations treated with calcium ionophore (A23187) exhibited increased production of PGs, TXB2, leukotriene (LT)B4, 5-HETE, 12-HETE, and 15-HETE, but the most striking increase occurred in metabolism through the lipoxygenase pathway. The major lipoxygenase metabolite generated by ionophore-stimulated macrophages was 5-HETE, and in intravascular macrophages 12-HETE was also produced. Preincubation of macrophages with indomethacin and nordihydroguaiaretic acid attenuated the yield of cyclooxygenase metabolites and lipoxygenase metabolites, respectively. Studies of leukotriene formation demonstrated that both macrophage types produce LTC4 and LTB4 from the leukotriene precursor LTA4. Thus, we show that the pulmonary intravascular macrophage is capable of metabolizing AA and LTA4 to their inflammatory and vasoactive metabolites by the cyclooxygenase and lipoxygenase pathways.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary alveolar proteinosis. Further evaluation of abnormal alveolar macrophages

To investigate the function of alveolar macrophages (AM) and the mechanisms of impairment in pulmonary alveolar proteinosis, we established in culture AM from three patients and from eight normal nonsmokers and assessed phagocytosis and phagolysosome fusion by the acridine orange assay with live yeast as the phagocytic challenge. Alveolar macrophages from the patients with pulmonary alveolar proteinosis ingested fewer yeasts per cell than did normal AM (mean +/- SE, 2.3 +/- 0.3 vs 3.3 +/- 0.2; p less than 0.05) and had decreased phagolysosome fusion (33 +/- 6 percent vs 64 +/- 1 percent; p less than 0.001). Alveolar macrophages from three normal subjects were incubated with cell-free fractions isolated by centrifugation of lavage fluid from the patients at 250 g (P1) or centrifugation of P1 supernatant at 20,000 g (P2). The P1 fraction did not decrease the number of AM ingesting yeast or the number of yeast cells ingested per cell, but the P2 fraction decreased both phagocytic indices. Conversely, phagolysosome fusion was depressed by the P1 fraction (48 +/- 3 percent vs 66 +/- 2 percent for untreated AM from the same subject; p less than 0.02) but not by the P2 fraction. Significant morphologic changes were noted in AM cocultured with both P1 and P2. Comparable concentrations of pooled P2 fractions from normal subjects did not decrease phagocytic indices in normal AM. These data confirm that AM in pulmonary alveolar proteinosis are dysfunctional, and, in particular, the finding of decreased phagolysosome fusion may be related to the high incidence of uncommon infections in these patients. We have shown that different fractions of alveolar filling material from patients with pulmonary alveolar proteinosis have unique effects on the phagocytic process in the normal AM, and the induced defects may be associated with apparent uptake of this material. These observations further support the hypothesis that in patients with pulmonary alveolar proteinosis, locally produced "toxic" substances may lead to impaired alveolar clearance and contribute to the pathogenesis of this disease.     

Tumor killing by human alveolar macrophages and blood monocytes. Decreased cytotoxicity of human alveolar macrophages

Tumor killing by human alveolar macrophages (AM) might be an important mechanism of pulmonary defense against neoplastic disease. We compared AM and blood monocytes (Mo) for the ability to kill 2 neoplastic targets, A549 human lung adenocarcinoma cells and P815 mastocytoma cells. Blood monocytes were able to kill both targets, whereas AM killed neither. Tumor killing by Mo was spontaneous and was not increased by incubation with lipopolysaccharide. Because the P815 target is highly sensitive to lysis by hydrogen peroxide (H2O2), it afforded the opportunity to compare AM and Mo for the ability to kill tumors by the production of toxic oxygen compounds. Comparable amounts of superoxide anion were produced by AM and Mo after stimulation with phorbol myristate acetate. However, luminol-enhanced chemiluminescence of AM was far less than that of Mo, suggesting that AM could not utilize the myeloperoxidase-H2O2-halide ion system for tumor killing. The addition of exogenous peroxidase to cultures of AM and P815 cells enabled AM to kill this tumor cell. Our results suggest that as Mo mature into AM, their ability to kill tumor cells declines and that AM may be unable to kill H2O2-sensitive tumors because of a loss of myeloperoxidase during maturation.