Augmentation of impaired tumoricidal function in alveolar macrophages from lung cancer patients by cocultivation with allogeneic, but not autologous lymphocytes

 It has been reported that the in vitro development of tumoricidal function in alveolar macrophages from lung cancer patients is reduced significantly when compared to that in peripheral blood monocytes from the same patients or alveolar macrophages from control patients. In the present investigation, a method for potentiating the development of tumoricidal function in alveolar macrophages from lung cancer patients is described. This method, which relies on priming the macrophages with purified, allogeneic peripheral blood lymphocytes from normal donors, could not be demonstrated when autologous lymphocytes from lung cancer patients were used in the priming coculture. The augmentation of tumoricidal function appears to be mediated by one or more soluble factors, since supernatants from cocultures of alveolar macrophages and allogeneic peripheral blood lymphocytes could enhance the cytotoxic function of freshly obtained alveolar macrophages. Furthermore, it appears that NK cells are necessary for this effect, since depletion of CD56⁺/CD57⁺ cells from allogeneic lymphocytes eliminated their capacity to enhance alveolar macrophage cytotoxic function. The augmentation of cytotoxic function elicited in alveolar macrophages by this method was not associated with changes in the secretion of tumor necrosis factor α, or interleukin 1β. Received: 15 March 1997 / Accepted: 11 June 1997     

Poly(I):poly(C)-enhanced alveolar and peritoneal macrophage phagocytosis: quantification by a new method utilizing fluorescent beads

Phagocytosis is an important immune function to quantify. This immune response may be modulated by exposure to biological response modifiers or by exposure to pollutants. A new technique for quantifying nonspecific phagocytosis of alveolar and peritoneal macrophages in the same animal has been developed that utilizes fluorescent polystyrene beads. When incorporated into inhalation studies, this technique can be used to determine whether the toxic effect of an inhaled pollutant is local (effect on alveolar macrophages), systemic (effect on peritoneal macrophages), or both local and systemic. This method results in a determination of both the level of phagocytosis (the percentage of phagocytic macrophages) and the macrophage specific activity (the number of beads phagocytized per macrophage). This method also allows a determination of adherence by quantifying the number of particles in contact with, but not phagocytized by, the macrophage. Macrophage preparations were incubated with fluorescent beads for 2 hr and cyto-centrifuged onto a glass slide. Fluorescent beads present on the slide or cell-associated but not ingested by phagocytosis were removed by immersing the slide containing the macrophage preparation in methylene chloride for 15-30 sec. Fluorescent beads ingested by phagocytosis were then easily quantified with a fluorescence microscope. This technique was used to assess the baseline levels of phagocytosis for rat alveolar and peritoneal macrophages from the same animal and the kinetics and level of enhanced phagocytosis for alveolar and peritoneal macrophages after injection with the interferon inducer polyinosinate-polycytidylate (poly(I):poly(C)). The kinetics of enhanced alveolar and peritoneal macrophage phagocytosis by poly(I):poly(C) were similar; however, stimulated phagocytic levels of peritoneal macrophages never reached the phagocytic activity observed for the resident, highly phagocytic alveolar macrophages. This elevated phagocytic activity is most likely due to interferon stimulated by particulate matter in the large volume of air processed by the lungs and is important for host defense against a number of different inhaled microorganisms.     

Effects of neonatal monosodium-L-glutamate treatment on rat alveolar macrophages

Treatment of neonatal rats with repeated doses of monosodium-L-glutamate resulted in changes of morphology and function of alveolar macrophages recovered from adult female rats. Numerous cellular lipid vacuoles and lamellar structures were observed in these alveolar macrophages under transmission electron microscopy. There was approximately 50% increase of cellular total lipid content measured by Oil-Red-O staining and colorimetric method. The yeast phagocytosis and intracellular killing ability, as well as the inhibitory effect on the growth of tumor cells were reduced in alveolar macrophages from neonatal MSG-treated rats.     

Detection of Fc receptors for IgA on rat alveolar and peritoneal macrophages.

The presence of Fc receptors for IgA on alveolar macrophages was determined by rosette assay and immunogold labeling. IgA-mediated phagocytosis by alveolar macrophages was observed. Results of these assays were compared between rats receiving no treatment and those receiving long-term cortisone administration. Sheep erythrocytes coated with dextran and an IgA monoclonal antibody specific for the alpha 1,3 linkages of dextran bound to 16% of alveolar macrophages. However, peritoneal macrophages did not form rosettes with dextran-IgA-coated erythrocytes. Immunogold labeling by transmission electron microscopy revealed that most Fc receptors for IgA were found on the membrane of pseudopodia of activated alveolar macrophages. Long-term cortisone administration diminished the phagocytosis and phagocytic index of alveolar macrophages, thereby contributing to decreased host resistance to infection (e.g., Pneumocystis carinii pneumonia).     

Effect of detergent solutions upon distal lung phospholipids

Intratracheally injected acid detergent solutions induce in rabbits a predominantly bronchiolo-lobular process evolving in two steps, an epithelial and an epitheliomesenchymal one. When the acid detergent solution reached prominently the alveolar structures it determined a rapid release by exocytosis of phospholipid lamellated bodies from the large granular alveolocytes and the following engulfment by macrophages of released products (Eskenasy, 1982 b). Histochemically, the cells containing phospholipids increased in cellularized alveolar walls, the alveolar lining film appeared thickened and denser, and the accumulated free macrophages filled with phospholipid reactive granules (Landing et al. 's method). Glucose-6-phosphate-dehydrogenase activity is very intense in the cellularly thickened alveolar walls, in macrophages and in the newly formed (regenerated) bronchiolar cells. Lactate dehydrogenase activity becomes very intense in macrophages. These data complete the morphogenic events leading to the lobular pathology.     

In vitro study of gas effects on alveolar macrophages

Summary To evaluate the biological effects of gas pollutants on alveolar macrophages several in vitro systems ave been developed. We described here an original method of cell culture in aerobiosis, which permitted direct contact between the atmosphere and the target cells. We studied the long term (24 h) and short term (30 min) effects of NO₂ on alveolar macrophages. Our results demonstrated that exposure of alveolar macrophages to gas pollutants may be responsible for either cell injury or cell activation associated with the release of various bioactive mediators (superoxide anion, neutrophil chemotactic activity). Cell culture in aerobiosis opens new ways for the research on the biological effects of gas pollutants.Abbreviations AM alveolar macrophages - CL Chemiluminescence     

Architectonics of the surface of alveolar macrophages

The architectonics of the alveolar macrophage surface has been investigated in the raster electron microscope. The material is obtained by means of washing from the lungs of intact noninbred white rats and also 24 h after a single intragastric administration of a cancerogenic agent--nitrosodimethylamine (NDMA)--in a toxic dose (30 mg/kg). The alveolar macrophages are studied both as a suspension and also after 30 min of cultivation. The preparations are dried in the air and by the critical point method. When the latter method is used, the architectonics of the alveolar macrophage surface is much richer. Nevertheless, the former method also gives enough information. NDMA administration produces a damaging effect on the surface architectonics and on the character of the macrophages spreading over the glass. The morphological characteristics of the changes in the surface architectonics of the alveolar macrophages can be used to estimate the cytotoxic effect of different harmful factors of the environment.     

An immunohistochemical method for the detection of tumour necrosis factor alpha in cytospins of human bronchoalveolar lavage cells

Summary An immunohistochemical method for assessing the level of tumour necrosis factor-α in alveolar macrophages obtained by brochoalveolar lavage is described. Cytospins of mixed populations of lung cells were incubated first with a monoclonal antibody to CD68 and then with a specific peroxidase-labelled second antibody in a two-step reaction for the detection of the macrophage marker CD68. A second similarly based two-step reaction for the detection of tumour necrosis factor-α followed. Both reactions were visualized, on completion, using different coloured peroxidase substrates which produced a third colour in the event of dual deposition of the substrates. Dual substrate deposition was indicative of alveolar macrophages positive for tumour necrosis factor-α. This method has provided a specific and reproducible semi-quantitative test for the presence of tumour necrosis factor-α in human activated alveolar macrophages, which can be performed retrospectively on clinical material. A range of concentrations of the cytokine has been demonstrated in individual samples. This dual detection method has the potential for detection of any cell-associated protein product by minor modification of the described method.     

Cystic fibrosis transmembrane conductance regulator-independent phagosomal acidification in macrophages

It was reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) is required for acidification of phagosomes in alveolar macrophages (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Here we determined whether the CFTR chloride channel is a generalized pathway for chloride entry into phagosomes in macrophages and whether mutations in CFTR could contribute to alveolar macrophage dysfunction. The pH of mature phagolysosomes in macrophages was measured by fluorescence ratio imaging using a zymosan conjugate containing Oregon Green(R) 488 and tetramethylrhodamine. Acidification of phagolysosomes in J774A.1 macrophages (pH approximately 5.1 at 45 min), murine alveolar macrophages (pH approximately 5.3), and human alveolar macrophages (pH approximately 5.3) was insensitive to CFTR inhibition by the thiazolidinone CFTR(inh)-172. Acidification of phagolysosomes in alveolar macrophages isolated from mice homozygous for DeltaF508-CFTR, the most common mutation in cystic fibrosis, was not different compared with that in alveolar macrophages isolated from wild-type mice. We also measured the kinetics of phagosomal acidification in J774A.1 and murine alveolar macrophages using a zymosan conjugate containing fluorescein and tetramethylrhodamine. Phagosomal acidification began within 3 min of zymosan binding and was complete within approximately 15 min of internalization. The rate of phagosomal acidification in J774A.1 cells was not slowed by CFTR(inh)-172 and was not different in alveolar macrophages from wild-type versus DeltaF508-CFTR mice. Our data indicate that phagolysosomal acidification in macrophages is not dependent on CFTR channel activity and do not support a proposed mechanism for cystic fibrosis lung disease involving defective phagosomal acidification and bacterial killing in alveolar macrophages.     

Binding of Griffonia simplicifolia I lectin to rat pulmonary alveolar macrophages and its use in purifying type II alveolar epithelial cells

We report that the isolectin Griffonia simplicifolia I-B4 isolated from G. simplicifolia seeds binds to rat alveolar macrophages present in frozen sections of lung tissue or bronchoalveolar lavage fluid. G. simplicifolia I-B4 does not bind to alveolar epithelial cells. We established that G. simplicifolia I-B4 binds to the macrophages via interaction with terminal alpha-D-galactopyranosyl residues present on these cells. This was substantiated by demonstrating that binding is inhibited either by the haptenic sugar alpha-D-galactopyranoside or by treating the cells with coffee bean alpha-galactosidase. Because murine laminin is known to contain terminal alpha-D-galactopyranosyl end-groups, and because we found that an anti-laminin antiserum binds to rat alveolar macrophages, we suspect that G. simplicifolia I-B4 may be binding to laminin present on the macrophages. To isolate alveolar type II epithelial cells from rat lungs, we developed a method that utilizes the lectin G. simplicifolia I. When proteinase-derived suspensions of pulmonary cells are incubated with G. simplicifolia I, the macrophages agglutinate and can be removed by filtration through nylon mesh. After incubating the resulting cellular suspension in tissue culture, the adherent cells are 94 +/- 2% (S.D.) type II cells. When compared to cells isolated by repeated differential adherence, the lectin-prepared type II cells have similar morphology and staining characteristics, form domes in monolayers and incorporate similar amounts of palmitate into disaturated phosphatidylcholine. We believe that the procedure outlined in this report provides a simple and effective method to isolate type II alveolar epithelial cells from rat lungs.     

Suppression of alveolar macrophage apoptosis prolongs survival of rats and mice with pneumocystis pneumonia

The number of alveolar macrophages is decreased in patients or animals with Pneumocystis pneumonia (Pcp). This loss of alveolar macrophages is in part due to apoptosis caused by Pneumocystis infection. The mechanism of apoptosis induction is unknown. Cell-free bronchoalveolar lavage fluids from Pneumocystis-infected rats or mice have the ability to induce apoptosis in normal alveolar macrophages. To characterize the mechanisms by which apoptosis proceeds in alveolar macrophages during Pcp, specific caspase inhibitors are tested for their ability to suppress the apoptosis. In vitro induction of apoptosis can be inhibited by the caspase-9 inhibitor (Z-LEHD-FMK) but not by the inhibitor to caspase-8 or -10. The caspase-9 inhibitor can also inhibit apoptosis of alveolar macrophages in vivo when it is intranasally instilled into dexamethasone-immunosuppressed, Pneumocystis-infected rats or L3T4 cell-depleted, Pneumocystis-infected mice. The number of alveolar macrophages rebounds in caspase-9 inhibitor-treated Pcp animals. Phagocytic activity of alveolar macrophages in treated animals is also recovered, and organism burden in these animals is reduced. Administration of caspase-9 inhibitor also clears the exudate that normally fills the alveoli during Pcp and decreases lung inflammation. Furthermore, caspase-9-treated Pcp animals survive for the entire 70-day period of the study, whereas nontreated Pcp animals die 40-60 days after initiation of infection. Depletion of recovered alveolar macrophages by intranasal administration of clodronate-containing liposomes in caspase-9 inhibitor-treated animals abrogates the effects of the inhibitor. Together, these results indicate that immunomodulation of the host response may be an alternative to current treatments for Pcp.     

Proteome alterations in primary human alveolar macrophages in response to influenza a virus infection

To obtain a global picture of how alveolar macrophages respond to influenza A virus (IAV) infection, we used a quantitative proteomics method to systematically examine protein expression in the IAV-infected primary human alveolar macrophages. Of the 1214 proteins identified, 43 were significantly up-regulated and 63 significantly down-regulated at >95% confidence. The expression of an array of interferon (IFN)-induced proteins was significantly increased in the IAV-infected macrophages. The protein with the greatest expression increase was ISG15, an IFN-induced protein that has been shown to play an important role in antiviral defense. Concomitantly, quantitative real-time PCR analysis revealed that the gene expression of type I IFNs increased substantially following virus infection. Our results are consistent with the notion that type I IFNs play a vital role in the response of human alveolar macrophages to IAV infection. In addition to the IFN-mediated responses, inflammatory response, apoptosis, and redox state rebalancing appeared also to be major pathways that were affected by IAV infection. Furthermore, our data suggest that alveolar macrophages may play a crucial role in regenerating alveolar epithelium during IAV infection.     

Production and secretion of adrenomedullin in cultured human alveolar macrophages.

To explore the role of adrenomedullin (ADM) in macrophages, we investigated the secretion of ADM by alveolar macrophages. Human alveolar macrophages obtained from bronchoalveolar lavage were cultured for 24 h. Northern blot analysis revealed ADM mRNA expression in alveolar macrophages. The levels of immunoreactive ADM in the media were 0.89+/-0.12 fmol/10(5) cells/24 h (n = 10). Reverse-phase high-performance liquid chromatography of the extract of culture media showed one major peak eluting in the position of the human ADM standard. The present study shows that alveolar macrophages produce and secrete ADM.     

Comparison of protein-synthesis rate of alveolar macrophages in vivo and in vitro.

This paper describes and validates a novel method for measuring rates of protein synthesis of rabbit alveolar macrophages in vivo. A rate of 9.3%/day was obtained, compared with 48.9%/day measured in vitro. This study suggests that the procedures involved in the isolation of alveolar macrophages for study in vitro may themselves activate the cell.     

Cooperative prosurvival activity by ERK and Akt in human alveolar macrophages is dependent on high levels of acid ceramidase activity

Human alveolar macrophages are unique in that they have an extended life span in contrast to precursor monocytes. In evaluating the role of sphingolipids in alveolar macrophage survival, we found high levels of sphingosine, but not sphingosine-1-phosphate. Sphingosine is generated by the action of ceramidase(s) on ceramide, and alveolar macrophages have high constitutive levels of acid ceramidase mRNA, protein, and activity. The high levels of acid ceramidase were specific to alveolar macrophages, because there was little ceramidase protein or activity (or sphingosine) in monocytes from matching donors. In evaluating prolonged survival of alveolar macrophages, we observed a requirement for constitutive activity of ERK MAPK and the PI3K downstream effector Akt. Blocking acid ceramidase but not sphingosine kinase activity in alveolar macrophages led to decreased ERK and Akt activity and induction of cell death. These studies suggest an important role for sphingolipids in prolonging survival of human alveolar macrophages via distinct survival pathways.     

Increased superoxide radicals generation from alveolar macrophages in immature guinea-pigs

To study the effect of maturation on abilities of superoxide radicals (O-2) generation in the airways, we compared stimuli-induced O-2 generation by alveolar macrophages in immature (aged 10+/-2 days) and adult (aged 90+/-2 days) guinea-pigs. The production of O-2 was assayed by chemiluminescence method, using a Cypridina luciferin analog as a highly sensitive and specific probe for O-2. Whereas no significant difference in cell components of bronchoalveolar lavage fluid was observed between immature and adult animals, O-2 generation induced by stimulation of alveolar macrophages was greater in immature than in adult animals, with significant differences observed after platelet-activating factor (100 nM) or phorbol myristate acetate (0.5 micro g/ml). The results suggest that alveolar macrophages from immature animals are far more potent O-2 generators than the same cells of adult animals.     

Effects of nickel oxide on the production of tumor necrosis factor by alveolar macrophages of rats

To evaluate the effect of green nickel oxide (NiO) on the production of tumor necrosis factor (TNF) by alveolar macrophages, alveolar macrophages were exposed to NiO in vitro and in vivo. For the in vitro study, rats alveolar macrophages were incubated with NiO on a microplate for 24 h. TNF activity in the culture supernatant was determined by the L929 bioassay. Rats alveolar macrophages cultured with 100 and 200 μg/mL of NiO in vitro induced the production of TNF, however, it was not statistically significant compared with the control that was free from NiO exposure. For exposure in vivo, rats were divided into two groups. Five were exposed to a daily concentration of 11.7±2.0 mg/m³ of NiO for an 8-hr/d, 5 d/wk, for 4 wk, and five rats (control) were kept in a cage and not exposed to NiO. Bronchoalveolar lavage was performed and the recovered alveolar macrophages were incubated on a microplate for 24 h. TNF production by exposed alveolar macrophages was significantly higher than that of controls.     

Lipopolysaccharide priming of alveolar macrophages for enhanced synthesis of prostanoids involves induction of a novel prostaglandin H synthase.

We report here that lipopolysaccharide (LPS) priming of rabbit alveolar macrophages leads to amplified synthesis of prostanoids, at least in part, by induction of a novel prostaglandin H synthase (PGH synthase). Rabbit alveolar macrophages were cultured with or without added LPS derived from Escherichia coli 0111:B4 for 4 h and then stimulated with opsonized zymosan (OPZ). LPS priming of alveolar macrophages resulted in enhanced release of thromboxane (TX) upon stimulation with OPZ, when compared to stimulated non-LPS controls. Addition of exogenous arachidonic acid to LPS-primed alveolar macrophages also resulted in increased production of TX. The LPS-induced increase in TX formation, in response to OPZ or arachidonic acid, was abolished by the addition of actinomycin D or cycloheximide during the priming period. Gas chromatography/mass spectrometry analysis indicated that levels of prostaglandins D2, E2, and F2 alpha, along with TX, were augmented in stimulated LPS-primed alveolar macrophages, implicating PGH synthase in the priming process. PGH synthase enzymatic activity, as determined by addition of arachidonic acid to macrophage sonicates, was markedly enhanced in LPS-primed alveolar macrophages. This correlated with increased PGH synthase levels detected by immunoprecipitation of 35S-labeled proteins and by Western blot analysis. Finally, Northern blot analysis using a cDNA probe to the recently described mitogen-inducible mouse PGH synthase revealed strong induction of approximately 4.3-kilobase mRNA in LPS-primed alveolar macrophages. Taken together, these results reveal that induction of a novel PGH synthase, probably the rabbit homologue of PGH synthase-2, plays a role in the enhanced synthesis of prostanoids by LPS-primed alveolar macrophages.     

Characterization of mononuclear phagocyte subpopulations in the human lung by using monoclonal antibodies: changes in alveolar macrophage phenotype associated with pulmonary sarcoidosis

Current concepts of pulmonary sarcoidosis suggest that the alveolar macrophage plays a central role in the pathogenesis of the disease. To help define the population of alveolar macrophages in sarcoidosis, we compared the surface phenotype of alveolar macrophages from patients with sarcoidosis and from normal individuals by using monoclonal antibodies (63D3, OKM1, M phi P-9, M phi S-1, 61D3, and M phi S-39) that detect surface antigens on cells of monocyte/macrophage lineage. Although almost all blood monocytes expressed surface antigens detected by each of these antibodies, only a minority of normal alveolar macrophages expressed the same surface antigens (p less than 0.05, each comparison). However, in sarcoidosis, the percentage of alveolar macrophages expressing these surface antigens was increased (p less than 0.05, each comparison with normal alveolar macrophages). Several findings supported the conclusion that the increased expression of these monocyte-lineage surface antigens on sarcoid alveolar macrophages resulted from increased recruitment of monocytes to the lung in sarcoidosis and not from abnormal "activation" of alveolar macrophages. First, alveolar macrophages expressing these antigens had an immature morphology. Second, in vitro cultivation of blood monocytes and alveolar macrophages in the presence of immune and inflammatory mediators, including mediators known to be present in the lung in sarcoidosis, did not prevent the loss of expression of monocyte-lineage surface antigens from monocytes or induce reexpression of monocyte-lineage surface antigens on alveolar macrophages. Third, the expression of monocyte-lineage surface antigens was only increased on sarcoid macrophages from patients whose lower respiratory tract contained an increased number of T lymphocytes, cells known to release monocyte chemotactic factor in sarcoidosis. Consistent with the knowledge that corticosteroids usually suppress the alveolitis of active sarcoidosis, when the expression of alveolar macrophage surface antigens was evaluated before and during therapy, the percentage of alveolar macrophages expressing monocyte-lineage surface antigens returned to normal after 1 to 3 mo of therapy.(ABSTRACT TRUNCATED AT 400 WORDS)