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Cell-free supernatant fluids obtained from BCG-sensitized lymph node cells (6 × 106cell/ml) incubated in tissue culture flasks containing heat-killed BCG (5 μg/ml) induced extensive development of multinucleated giant cells when incubated with normal alveolar macrophages. In contrast, supernatant fluids obtained after incubation of similar samples of the same lymph node cell population in flasks with (i) no mycobacteria, (ii) heat-killed Escherichia coli, or (iii) heat-killed Bacillus subtilisfailed to produce giant cells when added to normal alveolar macrophages. Giant-cell formation was observed in experiments using Nocardia brasiliensisas the eliciting antigen, indicating cross-reactivity between the antigens of this organism and BCG. These experiments indicate that BCG-sensitized lymphoid cells produce a soluble macrophage fusion factor after specific antigen stimulation. The fusion factor is a nondialyzable substance which is resistant to heating at 80 C for 30 min.

The responsiveness of alveolar macrophages lavaged from healthy volunteers to migration inhibitory factor (MIF) was tested by using the capillary-tube assay method. In every instance, macrophages from nonsmokers responded to MIF as demonstrated by a depression in migration of at least 30%, whereas MIF did not inhibit migration of macrophages from smokers. Cells from smokers migrated at a rate three times faster than cells from nonsmokers. Migration of macrophages from nonsmokers with delayed hypersensitivity skin reactions to Candida albicansantigen was inhibited when antigen was present in the tissue culture medium. Antigen did not inhibit macrophages from subjects who lacked delayed hypersensitivity to the antigen, or from subjects who were cigarette smokers. Since alveolar macrophages can respond to MIF in vitro, they may play a role in cell-mediated immune reactions in the lung. Cigarette smoking may interfere with this participation.

A number of noxious agents have been shown to affect the function and numbers of pulmonary alveolar macrophages. A protective factor which inhibits the lysis of alveolar macrophages was found in fluid lavaged from normal rabbit lungs. The protective action is lost following the in vitro and in vivo action of ozone. Dipalmitoyl lecithin exhibits a similar protective action, which is also lost following the in vitro action of ozone. The surface tension properties of the protective factor and dipalmitoyl lecithin may be altered, but not destroyed, following exposure to ozone. It is hypothesized that many environmental stresses, including ozone, may act by primary inhibition of a protective factor in lung fluid, which results secondarily in lysis of alveolar macrophages with loss of this defense mechanism of the lung against inhaled microorganisms and particulate matter.

The pulmonary alveolar macrophages possess metabolic pathways operative predominantly in the aerobic environment. These cells consume 0.15 to 0.2μmolar O2/mg protein/sec in vitro. Mitochondria from these cells manifest a respiratory rate of 0.5 to 0.6μmolar O2/mg protein/sec for succinate as a substrate. Mitochondrial oxidation is coupled to phosphorylation; adenosine diphosphate (ADP):oxygen (0) ratios are approximately 2 for flavin-linked and 3 for pyridine nucleotide-linked substrates. Cadmium ion adversely affects respiration of alveolar macrophages. It completely inhibits macrophage mitochondrial oxygen uptake at 50μmolar concentrations and uncouples oxidative phosphorylation at 5μmolar concentrations. This and several other divalent cations, like Cu++, Hg++, Sn++ and Zn++, also inhibit adenosine triphosphatase (ATPase) activity of alveolar macrophages. Since metal fumes and oxides are common air pollutants, a study of this sort might provide information on mechanisms at the biochemical level as to how toxic inhalants initiate pulmonary pathology.

Compared to cells from normal rabbit lungs, BCG-induced alveolar macrophages have a marked increase in hydrolase levels and the number of large electron-dense subcellular structures. This study was performed to investigate the possibility that these electron-dense structures were responsible for the increased hydrolase levels of these cells. Using sucrose gradient centrifugation, nuclei-free homogenates of normal and BCG-induced macrophages were analyzed with respect to the subcellular particles they contain. Gradient fractions were assayed for enzymes commonly associated with lysosomes as well as the mitochondrial cytochrome oxidase. Fractions of peak hydrolase activity from the BCG-induced preparations were consistently more dense than those from the normal cell preparations. Ultrastructural studies of the particulate material found in fractions of peak hydrolase activity from BCG-induced preparations revealed the presence of electron-dense, often dumbbell-shaped, granules. The data suggest that these peculiar granules are responsible for the elevated hydrolase levels of BCG-induced alveolar macrophages.

Nutter, J. E. (Fort Detrick, Frederick, Md.), and Q. N. Myrvik. In vitro interactions between rabbit alveolar macrophages and Pasteurella tularensis. J. Bacteriol. 92:645–651. 1966.—Rabbit alveolar macrophages were successfully employed in a study of host cell-Pasteurella tularensisinteractions in vitro. Under cell culture conditions in which inhibitory antibiotics were not employed and small infection ratios were used, the relative in vivo virulence of two strains of P. tularensiswas duplicated. As a consequence of intracellular multiplication, normal macrophages were killed in relation to the virulence of the strain employed. Alveolar macrophages were also collected from immune rabbits, and macrophage mortality and bacterial growth were significantly suppressed below levels observed with normal macrophage preparations. The effect of immune serum could only be ascribed a minor role in the observed reactions. A marked intravenous toxicity of P. tularensisfor the rabbit was observed with both virulent and attenuated strains. The toxicity was possessed only by viable preparations and could be elicited in animals immune to virulent challenge.

Acton, JeanD. (Bowman Gray School of Medicine, Winston-Salem, N.C.), and Quentin N. Myrvik. Production of interferon by alveolar macrophages. J. Bacteriol. 91:2300–2304. 1966.—Rabbit alveolar macrophages inoculated with parainfluenza-3 virus in vitro produce a viral inhibitor which possesses the properties of interferon. The interferon is nondialyzable, is stable at pH 4, is not sedimented at 100,000 × g, exhibits species specificity, and can passively protect other alveolar macrophages from infection with virulent rabbitpox virus. The possible significance of alveolar macrophage-produced interferon is discussed.

SummaryA technique was developed which provided large numbers of viable pulmonary alveolar macrophages from the mouse.These cells were labelled in vitro with 198Au and their distribution following intravenous injection in isologous strain mice was determined and compared with that of peritoneal macrophages in the same experimental system.Those transfused cells which localised in the liver were found to be indistinguishable both morphologically and functionally from indigenous Kupffer cells.In general, pulmonary alveolar and peritoneal macrophages were distributed in a very similar manner, although alveolar macrophages did not accumulate in the spleen in as high a concentration as did peritoneal macrophages.Australian Journal of Experimental Biology and Medical Science (1966) 44, 629–638; doi:10.1038/icb.1966.61

This investigation attempted to determine whether the primary source of alveolar macrophages is pulmonary or hematopoietic. We have utilized an antigenic marker to identify cells of hematopoietic origin. Mouse chimeras were produced by irradiating C57B6/AF1 mice (900 R) and then injecting them intravenously with B10D2/AF1 bone marrow. The donor animal has an antigenic specificity on the H-2 locus, not shared by the recipient. Alveolar macrophages were obtained by repeated lung washings with physiologic saline at 37°C. Cytotoxic tests were done on bone marrow and alveolar macrophages using anti-31 mouse antibody, absorbed rabbit serum as complement, and trypan blue exclusion as a test for viability. Animals were studied at 7, 14, 21, 28, and 35–50 days and 4, 5, 8, and 11 months after irradiation and bone marrow replacement. By 21 days after irradiation, 90% of the animals had greater than 80% replacement of marrow with donor tissue; and white blood cell and alveolar macrophage counts approached normal. At this time and at later intervals the per cent of donor cells in the lung free cell population was not significantly different from the per cent of donor cells in the bone marrow. Similarly, after aerosol particulate exposure, the percentage of marrow cells and alveolar macrophages of donor origin were not significantly different. This immunologic approach suggests that alveolar macrophages in radiation chimeras are entirely of hematopoietic origin.

A single intravenous injection of 0.1 mg of heat-killed Bacillus Calmette Guérin (BCG) in 0.1 ml of Bayol F produced an accumulation of activated alveolar macrophages (BCG induced). Cells were collected 3.5–4.0 wk after injection. Phospholipases A and three lysosomal marker enzymes (acid phosphatase, ß-glucuronidase, and lysozyme) were measured in homogenates, and the distribution of the phospholipases A and lysosomal, mitochondrial, and microsomal marker enzymes were examined after sucrose gradient centrifugation of a postnuclear (1,000 g) supernatant. Homogenates of normal and BCG-induced macrophages contained phospholipases A1 and A2 which had optimal activity at pH 4.0 in the presence of 2.0 mM ethylenediaminetetraacetate (EDTA). These activities were inhibited 50–70% by 2.0 mM CaCl2. Homogenates of BCG-induced macrophages had specific activities of ß-glucuronidase, acid phosphatase, and lysozyme, which were increased 1.5- to 3.0-fold over the controls, whether expressed as activity per mg protein or activity per 107 cells. The specific activities of the phospholipases A, on the other hand, were consistently lower than those of the control. Distribution of the phospholipases A and the lysosomal marker enzymes after sucrose gradient centrifugation suggested that the phospholipases A active at pH 4.0 in the presence of EDTA are of lysosomal origin since: (a) BCG treatment caused a selective increase in the density of particles which contained both the phospholipases A and three lysosomal marker enzymes; and (b) since the density of mitochondria and microsomes were not affected by BCG treatment. The increase in the density of lysosomes seen here may be related to previously described morphologic changes of BCG-induced alveolar macrophages.

The effects of temperature on two membrane transport systems in alveolar macrophages and one system in polymorphonuclear leukocytes which have been well characterized at 37° were analyzed. The results show a discontinuity in the Arrhenius plots (transition temperature) for both the alveolar macrophage systems but none in the polymorphonuclear leukocytes. A detailed analysis of the specificity of nucleoside transport in alveolar macrophages reveals a marked temperature dependence which is reflected mainly in requirements at the 3′ (ribose)-position. Pretreatment of the alveolar macrophages with colchicine alkaloids results in a transport system for nucleosides which shows no transition temperature and has the specificity and activation energy of the untreated system in the low temperature range. On the basis of these findings it is proposed that the nucleoside carrier exists as two or more conformers, one of which is favored by high temperatures, and the other by low temperatures or colchicine alkaloids.

Rabbit alveolar macrophages can induce the L-phase of Listeria monocytogenesin vitro. Microscopic examination of Listeria-infected cultures revealed the presence of intracellular wall defective variants. Isolation techniques using saran membranes yielded L-phase variants which exhibited typical L-form colonies on osmotically protected medium. All attempts at reverting the isolated L-phase have been unsuccessful.

The acid phosphatase activity of normal alveolar and BCG-induced alveolar macrophages has been examined. Five electrophoretically distinct forms of acid phosphatase have been identified in both normal and BCG-induced macrophages. The acid phosphatases can be divided into two major categories. One category, containing four distinct forms, is readily solubilized after repeated freezing and thawing or mechanical disruption The second category, containing one form, is firmly bound to the lysosomal membrane and can be solubilized by treatment of the lysosomal fraction with Triton X-100. The Triton-extractable acid phosphatase and the predominant aqueous soluble acid phosphatase have been shown to differ in the degree of membrane binding, in solubility, in net charge, and in molecular weight. The two pre-dominant phosphatases possess identical pH optimum and do not differ in response to enzyme inhibitors. BCG stimulation has been shown to result in a nearly twofold increase in acid phosphatase activity. A nearly proportionate increase in the major acid phosphatase forms has been observed.

Pulmonary alveolar macrophages (PAMs) constitute a major lung cellular defense mechanism. There is still considerable controversy concerning the cellular precursors of the resident PAM population. Genetically related (RF/AI( + ) and RF/AI(-) substrains of mice were used to investigate the possibility of PAM origin from marrow stem cells. The RF/AI( +) mice carry a nonspecific readily identifiable prealbumin esterase marker, Es-2. Reciprocal chimerism was produced by administering a lethal dose of x-irradiation to (1) RF/AI(-) mice and subsequently injecting them with RF/AI( + ) bone marrow, and (2) RF/ Al( +) mice and subsequently injecting them with RF/AI(-) bone marrow. Eight weeks after irradiation pulmonary macrophages were obtained by bronchial lavage. PAMs from RF/AI(-) animals transplanted with RF/AI( +) bone marrow showed the prealbumin esterase marker, Es-2, while PAMs from the RF/AI( + ) animals given RF/AI(-) bone marrow had all but lost their Es-2 marker band, which confirms the marrow origin of PAMs.

The effects of phagocytosis on the metabolic responses of rabbit alveolar macrophages have been studied in vitro. These cells exhibit peroxidative metabolism which is stimulated phagocytosis. One H2O2 generating oxidase, D-amino acid oxidase, has been demonstrated. Two H2O2 utilizing pathways exist, namely a nicotinamide adenine dinucleotide phospate (NADP) linked cytoplasmic glutathione shuttle and catalase-dependent reactions.

Previous studies have shown that ozone in low concentration in vivo depresses the activity of alveolar macrophage acid phosphatase, lysozyme, and β-glucuronidase. In the present studies, rabbit alveolar macrophages were held in tissue culture as a monolayer and were exposed to various concentrations of ozone when covered by only a film of tissue culture media. This approach eliminated recovery of a mixed population of cells from the lung and possible influx of macrophages in response to the noxious agent. Ozone in moderately low concentration, 2 ppm, depressed the activity of macrophage lysosomal acid hydrolases. There was slight accumulation of enzyme in extracellular media during and after three hour exposure to ozone. Hydrolase depression effect was eliminated when the culture media contained sulfhydryl donating reagents—glutathione and cysteine. This may offer a clue to a mechanism of ozone toxicity.

Rabbits sensitized intravenously with heat-killed Mycobacterium tuberculosis(strain H37Ra) suspended in mineral oil developed a strong pulmonary granulomatous response which reached its peak about 3 to 4 weeks after injection. Alveolar cells (4 × 106cells/ml of tissue culture medium 199) procured 6 weeks after sensitization showed extensive development of multinucleated giant cells after 12 hr of incubation in tissue culture flasks containing heat-killed H37Ra (5 μg/ml). Giant cells measured 80 μm to 2.5 mm in length and contained between 30 and 700 nuclei. In contrast, no giant cells were observed when similar samples of the same cell populations were incubated in flasks containing: (i) no mycobacteria; (ii) heat-killed Escherichia coli; (iii) heat-killed Bacillus subtilis; (iv) latex particles; (v) ovalbumin; or (vi) phytohemagglutinin. The addition of immune (anti-H37Ra) sera potentiated the phenomenon of giant cell formation. In addition, supernatant fluids obtained from sensitive alveolar cells incubated with H37Ra were capable of inducing giant cell formation when incubated with nonsensitized alveolar cells. The results suggest that fusion of alveolar macrophages is mediated by an immunological mechanism.