Cytostatic effect of immune system cells on tumor cells

The cytostatic effect of different lymphoid cells on tumour cells was studied in mice. It was shown that the cells differed in their ability to manifest cytostatic and natural cytotoxic activity. The interstrain variations of cytostatic activity levels in murine splenocytes were revealed. The degree of cytostatic action of effector cells on tumour cells depended both on effector-target cells ratio and on the incubation time of effector and target cells. The cytostatic effect of splenocytes and macrophages was demonstrated to be unrestricted by H-2 complex and independent of the tumour type. The data suggest that the cytostatic effector cells are heterogeneous and may be distinct from natural killer cells.     

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.     

Macrophage heterogeneity in tumor resistance: Cytostatic and cytotoxic activity of corynebacterium parvum-activated and proteose peptone-elicited rat macrophages against Moloney sarcoma tumor cells

Peritoneal macrophages from proteose peptone and Corynebacterium parvum (CP)-treated Lewis and Brown Norway rats were separated into subpopulations by centrifugation on discontinuous gradients of Ficoll. Four macrophage subpopulations were prepared and tested for cytostatic and cytotoxic activity against syngeneic and allogeneic Moloney sarcoma tumor cells. Macrophages were cocultured with tumor cells for 48 hr, whereupon either the inhibition of [¹²⁵I]iododeoxyuridine uptake was measured (cytostasis) or the tumor monolayers were observed for cytotoxic effects. CP-Activated macrophages from heavy-density portions of the gradient (8–10% and 10%-pellet) were highly cytostatic and cytotoxic to both the syngeneic and allogeneic tumor cells while macrophages from the light-density portions (4–6 and 6–8% Ficoll bands) were not. Proteose peptone-stimulated macrophages from the heavy-density portions of the gradient were cytostatic but not cytotoxic to the tumor cells. The effector macrophages from the CP-activated pool were large, well-differentiated cells as determined by electron microscopic examinations and had enhanced phagocytic activity when contrasted with the noncytotoxic, less dense macrophages.     

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     

Respiratory burst in alveolar macrophages of diabetic rats

Bactericidal ability of alveolar macrophages is depressed in rats with diabetes mellitus. To define the mechanism of this abnormality, we measured the parameters of respiratory burst in alveolar macrophages, peripheral blood monocytes, and neutrophils of rats 8 wk after the induction of diabetes by streptozocin. Superoxide anion (O2-.) generation during basal conditions and after stimulation with phorbol myristate acetate (PMA) was measured as superoxide dismutase-inhibitable cytochrome c reduction. NADPH, the principal substrate for NADPH-oxidase-dependent O2-. generation, was measured in the alveolar macrophages and quick-frozen lungs by the enzyme-cycling method. O2-. generation after PMA was significantly lower in the alveolar macrophages of diabetics than in the controls (14.4 +/- 2.0 nmol.10(6) cells-1.20 min-1 vs. 26.2 +/- 1.9, P less than 0.05). Conversely the peripheral blood monocytes of diabetics demonstrated an enhanced O2-. production after PMA stimulation. There was no significant difference in the neutrophil O2-.-generation between the groups. The alveolar macrophage NADPH (control 0.44 +/- 0.15 nmol/10(6) cells vs. diabetic 0.21 +/- 0.04, P less than 0.05) and lung tissue NADPH levels (control 81.4 +/- 16.3 nmol/g dry wt vs. diabetic 35.8 +/- 20.5, P less than 0.05) were significantly lower in the diabetics than in the controls. These data indicate that the O2-.-generating capacity of alveolar macrophages is markedly depressed in diabetes, whereas their precursors, monocytes, are primed to generate O2-. with PMA stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of activated macrophages on tumor target cells: Escape from cytostasis

In contrast to normal mouse peritoneal macrophages, activated macrophages almost totally inhibit [³H]TdR uptake by tumor target cells 24 hr after challenge. However, when the period of observation was extended to 48 or 72 hr, renewed [³H]TdR uptake by target cells was often, but not always, observed in the presence of activated macrophages. This apparent escape of target cells from the cytostatic effects of activated macrophages was not due to a subpopulation of resistant target cells, and autoradiographic studies revealed that target cells, inhibited from incorporating [³H]TdR by activated macrophages at 24 hr, were subsequently able to renew DNA synthesis and multiply. These results suggest that in the presence of activated macrophages, the almost total cytostasis of target cells does not necessarily mean that these cells are irreversibly damaged or killed.Escape from or maintenance of cytostasis was not peculiar to any of the target cells (L cells, EMT-6, Bladder 4934) or mouse strains (SW, C57BL, BALB/c) employed nor was it consistent with any of the forms of stimulation used for obtaining activated macrophages (Toxoplasma or Besnoitia infection; C. parvum treatment). However, the results suggest that when escape of target cells from the cytostatic effects of activated macrophages occurred, it may have been due to a qualitative or quantitative inadequacy of the population of macrophages employed.     

Cytostatic and cytolytic activities of macrophages regulation by prostaglandins

Murine peritoneal macrophages (M phi), activated in vivo or in vitro, remarkably inhibited the uptake of thymidine by a lens epithelial cell line, while resident M phi, or M phi induced by thioglycollate, exhibited much lower or no cytostatic capacity. The target cells were partially protected from the cytostatic activity by the anti-inflammatory agents indomethacin, aspirin, and dexamethasone, but not by lipoxygenase inhibitors. The protective activity of indomethacin and aspirin, but not of dexamethasone, was completely counteracted by prostaglandin E2 (PGE2). Yet, PGE2 alone has no effect on the uptake of [3H]thymidine by lens epithelial cells. PGE1 resembled PGE2 in its effect on this system, whereas PGA2, PGB2, or PGF2 alpha had no detectable activity. The counteracting effect of PGE2 was mimicked by dibutyryl cAMP or by cholera toxin, an agent which increases cAMP levels. These findings suggest that PGEs are not direct cytostatic agents, but rather, are essential mediators for the development of the cytostasis. Activated M phi did not lyse cells of the original lens epithelial cell line, but caused substantial cytolysis of cells of a subline derived from it. In contrast to its aforementioned effect on the cytostasis, PGE2 inhibited the cytolytic activity of M phi. Thus, this study provides a first demonstration in a single system of the opposite effects of PGEs on M phi activity on target cells, i.e., mediating the cytostasis and inhibiting the cytolysis.     

Enhanced interleukin 1 production by alveolar macrophages and increase in Ia-positive lung cells in silica-exposed rats

Inhalation exposure to silica dust enhanced interleukin 1 (IL-1) production by alveolar macrophages (AM), which is attributable to an increase in Ia-positive lung cells. While the proportion of Ia-positive cells in lavaged bronchoalveolar cells (BAC) was much lower (0-3%) in unexposed control rats, about a third of the rats that inhaled silica showed higher proportions (8.0-18.5%); these were designated "Ia-high" exposed animals. The number of total cells, Ia-positive cells and lymphocytes in BAC was significantly increased (P less than 0.05, P less than 0.001, and P less than 0.001, respectively) in these "Ia-high" exposed animals, compared to the control animals. Adherent AM populations obtained from BAC preparations also contained significantly higher (P less than 0.001) proportions of Ia-positive cells in the "Ia-high" exposed animals. When these adherent AM cultures were stimulated with lipopolysaccharide, IL-1 activity of the culture supernatants was enhanced and was significantly higher (P less than 0.001) in the "Ia-high" exposed rats, compared to the control animals. These results indicate that silica-exposure can induce populational changes in lung cells and also activation of AM associated with the increase in Ia-positive cells.     

Cytotoxic effects of 3-methylindole on alveolar epithelial cells and macrophages: with special reference to microtubular and filamentous assemblies in alveolar type I cells of bovine lung

The alveolar type I cell is a major permeability barrier between the pulmonary interstitium and alveolar spaces and its thin cytoplasmic processes are greatly susceptible to injury. These cells are often observed to undergo progressive vesiculation, vacuolization and desquamation during 3-methylindole (3MI)-induced acute pulmonary edema after oral administration in goats and cattle. The present study describes proliferation of SER and the presence of polymerized tubulin in the form of microtubules arranged in large bundles shown at ultrastructural level as well as with immunofluorescence staining for tubulin in alveolar type I cells 72 hours after 3MI treatment. Such changes were not seen in pulmonary endothelial cells, alveolar type II cells, alveolar macrophages and neutrophils. The possible role of microtubules in alveolar type I cells as a mechanistic support to resist disruption against the forces of interstitial and alveolar edema is compared with alveolar type II cells, alveolar macrophages and neutrophils. The latter cells undergo dynamic movements in response to inflammatory stimuli and therefore did not show microtubules in their cytoplasm.     

Role of alveolar macrophages in endotoxin-induced neutrophilic alveolitis in rats

Although bacterial endotoxins have potent effects on blood monocytes and tissue macrophages, the role of alveolar macrophages in regulating intrapulmonary neutrophil traffic following endotoxemia has not been studied previously. We have previously reported that a single intraperitoneal injection of endotoxin from Escherichia coli serotype 055B5 causes acute lung inflammation by neutrophils (PMN) in rats. The factors which influence the migration of PMN in the lung in this model are unknown. To determine whether macrophage-derived products could play a role in directing migration, we enumerated neutrophils in histologic sections and employed electron microscopy to document the location of neutrophils in the lung in vivo following endotoxin. We also cultured the alveolar macrophages recovered by lung lavage to measure the effect of their culture supernatants on neutrophil migration in vitro. In the first 6 hr following endotoxin, and also 24 hr later, there was an increase in the number of PMN enumerated in the lung parenchyma by light microscopy. Electron microscopy showed the location of the neutrophils to be exclusively intravascular at 6 hr. By contrast, neutrophils were observed in both interstitial and bronchoalveolar spaces at 24 hr, confirming that transvascular migration was active at that time. The pulmonary macrophages which were recovered by lung lavage from groups of rats sacrificed at 4 and at 15 hr following the administration of endotoxin were assayed for the release into culture media of migration-stimulatory activity for neutrophils. Macrophages from animals sacrificed 4 hr following endotoxin released less migration-stimulating activity into media than macrophages from controls. These macrophages could be stimulated to release migration-stimulating activity into culture media at levels comparable to macrophages from controls by the addition of opsonized Zymosan to the culture media. By contrast, macrophages from animals sacrificed 15 hr after endotoxin spontaneously released more migration-stimulating activity for neutrophils than did macrophages from controls. Thus, in this model, a specific increase in the synthesis or release by alveolar macrophages of factors which stimulate the migration of neutrophils in vitro coincided with a transition from intravascular to extravascular alveolar inflammation by neutrophils in vivo. These observations are consistent with the hypothesis that pulmonary alveolar macrophages may contribute to the regulation of alveolar inflammation following endotoxemia by releasing factors which influence the migration of neutrophils.     

Taurine uptake by isolated alveolar macrophages and type II cells

Evidence suggests that taurine may protect cellular membranes against oxidants (Gordon et al., Am. J. Pathol. 125: 585-600, 1986). The present study was conducted to determine if alveolar macrophages and type II cells (which are relatively resistant to oxidant injury) possess a specialized transport system for the accumulation of taurine. The results indicate that both cell types contain more taurine than plasma or whole lung. Taurine influx exhibited both carrier-mediated and simple diffusion components. Carrier-mediated uptake displayed saturation kinetics (Km = 26.3 and 22.5 microM, while Vmax = 33.2 and 4.9 pmol.10(6) cells-1.min-1 for macrophages and type II cells, respectively). Taurine uptake was dependent on extracellular sodium and inhibited by metabolic inhibitors or ouabain. Total taurine uptake by type II cells was lower than that of alveolar macrophages. However, type II cells exhibited a higher intercellular concentration of taurine (14 vs. 4 mM) because of a higher ratio of carrier-mediated uptake to leakage than with alveolar macrophages. It is possible that this specialized transport system for taurine uptake may lend these cells resistant to oxidant injury.     

Protective effects of green tea catechins on alveolar macrophages against bacterial infections

Bacterial pneumonia in immunocompromised patients as well as elderly persons often becomes a life threatening disease, even when effective antibiotics are used extensively. In addition, the appearance of antibiotic-resistant bacteria in medical facilities as well as in patients requires another approach to treat such patients besides treatment with antibiotics. In this regard, green tea catechins, such as epigallocatechin gallate (EGCg), may be one of the potential agents for such purpose due to its possible potential immunomodulatory as well as antimicrobial activity. The studies by us showed that EGCg enhanced the in vitro resistance of alveolar macrophages to Legionella pneumophila infection by selective immunomodulatory effects on cytokine formation. Furthermore, the tobacco smoking-induced impairment of alveolar macrophages regarding antibacterial as well as immune activity was also recovered by EGCg treatment. These results indicate that EGCg may be a possible potential immunotherapeutic agent against respiratory infections in immunocompromised patients, such as heavy smokers.     

Radiation effects on oxidative metabolism in young and aged rat alveolar macrophages.

The effect of ionizing radiation on metabolic functions of alveolar macrophages (AM) have been well studied. However, variations associated to age have not been established yet. The aim of this work was to perform a comparative study on irradiated alveolar macrophages from young and aged rat lungs. Cell viability and occurrence of apoptosis as well as production of nitric oxide (NO), generation of superoxide anion (O2*-) and total antioxidant capacity were analyzed in vitro after exposure to gamma-irradiation with 10, 25, 50 and 75 Gy. Cell viability decreased only in the aged population at the higher doses. Morphological features of apoptosis were clearly evidenced in irradiated alveolar macrophages from aged animals although the DNA fragmentation assay for apoptosis showed no differences for either of the populations studied. NO production and total reactive antioxidant potential (TRAP) levels showed a dose-dependent modulation. Low radiation doses inhibited the production of NO and decreased TRAP levels whereas higher doses enhanced the NO production and increased the TRAP levels in both macrophage populations. Generation of O2*- was always higher in the aged population for all the doses assayed. We conclude that in vitro young alveolar macrophages exhibited higher radioresistance over the whole range of doses as compared to the aged macrophage population. Our results show that the aging process markedly affects the radioresistance of phagocytic cells. Therefore, immune defense and inflammatory response of lungs from aged patients should be considered when planning radiotherapy protocols.     

The effects of silica dust and alveolar macrophages on lung fibroblasts grown in vitro

The effect of silica (min-u-sil) on lung fibroblasts, with and without the mediation of alveolar macrophages, has been studied by measuring DNA, protein, collagen, lysosomal enzymes and secreted glycosaminoglycans. Intact macrophages were found to stimulate collagen production whether they had first been pretreated with silica or not. The dust has a direct effect on fibroblasts, an effect dependent on silica concentration and the stage of fibroblast growth. The possible relationships of the above effects to both fibrosis and emphysema are discussed.     

Activation of antitumor properties in alveolar macrophages from protein-calorie malnourished rats

Summary Protein-calorie malnutrition (PCM) was induced by feeding male F344 rats on a 5% casein diet for 7 weeks. At appropriate times, rats from control (20% casein diet) and PCM groups were killed and alveolar macrophages (AM) were obtained by bronchoalveolar lavage. The functional integrity of the AM was determined by measuring their ability to become tumoricidal on treatment with macrophage activators, such as muramyl dipeptide (MDP) or multilamellar liposomes containing MDP or its lipophilic analog, MTP-PE. After 5 and 7 weeks, the numbers of lavaged AM per gram body weight of rats were much higher in the PCM group than in the control group. In week 3, AM from the PCM group showed spontaneous tumoricidal activity against syngeneic tumor cells, but in weeks 5 and 7 they did not. However, AM from PCM rats behaved the same way as controls in their response to activation stimuli in vitro with multilamellar liposomes containing synthetic MDP or MTP-PE.These data show that PCM affects the number of AM, but that AM from rats in a state of PCM become tumoricidal in response to activation stimuli in vitro.     

Pulmonary surfactant obtained from starved rats enhances phagocytosis of alveolar macrophages

Phagocytic activity of alveolar macrophages (AM) was enhanced by pulmonary surfactant obtained from bronchoalveolar lavage fluid of rats starved for 2 days, as compared to fed. The enhanced activity of phagocytosis was dependent on the dose of surfactant. The prepared surfactant showed a different protein to phospholipid ratio of 0.108 in fed and 0.234 in 2 days starved, because of an increased ratio of protein in surfactant from 2 days starved rats. F(ab')2 anti-surfactant protein inhibited the enhanced AM phagocytosis by surfactant. These results suggested that the enhancement of AM phagocytosis in 2 days starved rats was on account of an increase of protein in their surfactant compared to fed.     

Meconium enhances platelet-activating factor and tumor necrosis factor production by rat alveolar macrophages

Meconium aspiration syndrome (MAS) frequently results in inactivation of surfactant, persistent pulmonary hypertension (PPHN) and respiratory failure among newborn infants. Inflammation and inflammatory mediators play an important role in MAS. Since alveolar macrophages are thought to be very important cells in the pathogenesis of various inflammatory diseases, we evaluated whether meconium could stimulate rat alveolar macrophages to generate platelet-activating factor (PAF) and tumor necrosis factor (TNF)-alpha in vitro. We also examined the response to A23187 (calcium ionophore), 1-0-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (synthetic PAF) and dexamethasone on meconium-induced release of PAF and TNF-alpha. PAF and TNF-alpha concentrations from supernatant fluid were measured after high-performance liquid chromatography purification by specific radioimmunoassay, and TNF-alpha concentrations were determined by using an enzyme-linked immunosorbent assay. Our results showed that alveolar macrophages exposed to meconium could enhance PAF and TNF-alpha production in a dose (0.1, 1, 5 and 10%, P<0.01)-dependent way. In the presence of A23187, the capability of meconium to stimulate PAF production was further enhanced in the supernatant fluids. Furthermore, treatment with synthetic PAF significantly increased the generation of TNF-alpha in response to meconium. On the other hand, dexamethasone effectively inhibited both PAF and TNF-alpha production stimulated by 5% meconium (P<0.01, P<0.01; respectively). We suggest that alveolar macrophages and PAF, TNF-alpha play an important role in the pathogenesis of lung injury and severe complications in MAS. Furthermore, the protective effect of glucocorticoids in MAS could be due, at least in part, to a suppression of PAF and TNF-alpha generation.     

Ultrastructure of tumor cell interaction with alveolar macrophages stimulated by vitamin A

F344 rats were given vitamin A for four consecutive days and then their alveolar macrophages (AM phi) were obtained by bronchopulmonary lavage of the lung. Compared with unstimulated AM phi, AM phi from rats given vitamin A had more numerous and longer cytoplasmic projections, and these projections had many knobs on their sides and tip. The AM phi became attached to syngeneic mammary adenocarcinoma cells at many focal points and the tumor cells then lost surface microvilli around the contact zones. Detachment of the knobs from the projections on AM phi was often observed in areas of close association between AM phi and tumor cells. The detached knobs were 250 nm in diameter, gave a positive reaction for acid phosphatase, and frequently became attached to the surface of tumor cells. Then, many of the tumor cells in the vicinity of AM phi exhibited cytolytic changes. It is concluded that the cytotoxicity of stimulated AM phi is due to their attachment to the surface of tumor cells and their release of particles with acid phosphatase activity into the narrow space between the cells, and then to uptake of these particles by susceptible tumor cells.