Angeline E. Warner

Gadolinium induces macrophage apoptosis

Gadolinium (Gd) suppresses reticuloendothelial functions in vivo by unknown mechanisms. In vitro exposure of rat alveolar macrophages to GdCl3·6H2O caused cell death, as measured by trypan blue permeability, in both dose‐ and time‐dependent fashions. Even a 10‐min exposure to Gd caused significant cell death by 24 h. The morphology of Gd‐treated cells, pyknosis and karyorrhexis prior to loss of membrane integrity, suggested apoptosis. Upon flow cytometric examination, Gd‐treated propidium iodide‐excluding cells demonstrated light scatter changes characteristic of apoptotic cells (decreased forward and increased right angle scatter). Gel electrophoresis of DNA from Gd‐treated macrophages clearly showed the ladder pattern unique to apoptotic cells. Electron‐dense structures containing Gd were observed via electron spectroscopic imaging within phagosomes and also within nuclei (associated with condensed chromatin). Gadolinium, endocytosed by macrophages and distributed to nuclei, causes apoptosis of macrophages in vitro.

Pulmonary intravascular macrophages: their contribution to the mononuclear phagocyte system in 13 species

The organ uptake of intravenously injected particles was examined in 13 species. All animals were injected intravenously with 198Au colloid and magnetic iron oxide particles. Vascular clearance kinetics of 198Au colloid was similar in all species. Pulmonary uptake of 198Au colloid ranged from 17 to 60% in sheep, calves, pigs, and cats but was <1.1% in monkeys, hyraxes, rabbits, guinea pigs, rats, mice, and chickens. For iron oxide particles, pulmonary uptake ranged from 80 to 99% in sheep, calves, pigs, goats, and cats and 15 to 18% in hamsters, hyraxes, and monkeys and was <10% in rabbits, chicken, mice, rats, and guinea pigs. In all species, the bulk of the remainder of particle uptake was in the liver. Pulmonary intravascular macrophages are the cellular site of lung uptake in calves, cats, pigs, goats, and sheep, whereas monocytes and neutrophils predominate in other species. Kupffer cells were the site of uptake in the liver. Our data show marked species differences in the fate of circulating particles; ruminants, pigs, and cats have extensive pulmonary localization due to phagocytosis by pulmonary intravascular macrophages.The organ uptake of intravenously injected particles was examined in 13 species. All animals were injected intravenously with 198Au colloid and magnetic iron oxide particles. Vascular clearance kinetics of198Au colloid was similar in all species. Pulmonary uptake of 198Au colloid ranged from 17 to 60% in sheep, calves, pigs, and cats but was <1.1% in monkeys, hyraxes, rabbits, guinea pigs, rats, mice, and chickens. For iron oxide particles, pulmonary uptake ranged from 80 to 99% in sheep, calves, pigs, goats, and cats and 15 to 18% in hamsters, hyraxes, and monkeys and was <10% in rabbits, chicken, mice, rats, and guinea pigs. In all species, the bulk of the remainder of particle uptake was in the liver. Pulmonary intravascular macrophages are the cellular site of lung uptake in calves, cats, pigs, goats, and sheep, whereas monocytes and neutrophils predominate in other species. Kupffer cells were the site of uptake in the liver. Our data show marked species differences in the fate of circulating particles; ruminants, pigs, and cats have extensive pulmonary localization due to phagocytosis by pulmonary intravascular macrophages.

QUANTITATIVE RECOVERY OF PULMONARY INTRAVASCULAR MACROPHAGES FROM SHEEP LUNGS

Pulmonary intravascular macrophages (PIMs) adhere to the endothelium of lung capillaries and sequester circulating particles and pathogens from the blood. Iron oxide (γFe203) 5 mg/kg, administered intravenously, specifically labeled PIMs in situ within the living sheep. Attempts to isolate γFe203‐labeled PIMs using vascular perfusion (VP) procedures yielded few cells. To improve recovery of PIMs, a proteolytic lung digestion (PLD) procedure was developed. Following PLD, γFe203‐containing PIMs were recovered by magnets and the amount of γFe203 present measured by fluxgate magnetometry. Proteolytic lung digestion recovered 34% of the total γFe203 in lung samples and yielded 2 105 PIMs/g lung with 95% viability. In contrast, VP recovered only 3% of the total γFe203 in the lung; furthermore, less than 2% of the recovered γFe203 was cell associated. Proteolytic lung digestion followed by magnetic separation is an effective way to recover viable sheep PIMs for in vitro study. J. Leukoc. Biol. 56: 692–701; 1994.

The Effect of Endotoxin on in Vivo Rat Alveolar Macrophage Phagocytosis

This study was performed to explore whether alveolar macrophage (AM) phagocytosis would be impaired during endotoxemia. Therefore, we characterized in vivo AM phagocytic function in rats following either intravenous (i.v.) or intratracheal (i.t.) administration of lipopolysaccharide (LPS). The i.v. administration of LPS to rats at dosages of 0, 1, 2, and 5 mg/kg showed that increasing LPS doses were significantly associated with increased AM phagocytosis of 198Au colloid (P < .01), decreased recovery of AMs in bronchoalveolar lavage (BAL) (P = .017), no significant differences in neutrophil recovery by lavage (P = .15), or in the concentration of albumin in BAL (P = .14). Across the dosages of LPS administered i.t. (i.e., 0, 1, 5, and 10 mg/kg), there was no difference in AM phagocytosis (P = .29), a significant decrease in AM recovery (P = .002), a significant increase in neutrophil number (P = .01), and little effect on the concentration of albumin (P = .06). Thus, we found that the administration of endotoxin to rats did not impair in vivo AM phagocytic function. In fact, our findings suggest that the i.v. administration of LPS may increase AM phagocytosis of 198Au.

Effects of a perfluorochemical emulsion on the fate of circulating Pseudomonas aeruginosa.

Because mononuclear phagocytes take up perfluorochemical emulsions (PFCE), we examined how prior treatment with PFCE affects the fate of circulating bacteria. Rats were preinjected with three daily intravenous injections of PFCE (2.0 ml/100 g) containing 12.5% (vol/vol) of a 4:1 mixture of F-dimethyl adamantane and F-trimethylbicyclo-nonane, 2.5% (wt/vol) Pluronic F-68 as the emulsifying agent, and 3% (wt/vol) hydroxyethyl starch as the oncotic agent. Pseudomonas aeruginosa or Staphylococcus aureus were injected 4 h after the third PFCE injection. PFCE pretreatment decreased the rate and extent of vascular clearance of P. aeruginosa, with decreased uptake by the liver. Importantly, there were significant decreases in killing of P. aeruginosa in the liver, lungs, spleen, and kidneys of PFCE animals. PFCE did not alter the clearance of S. aureus from the circulation. However, hepatic uptake was reduced, with concomitant increases in lung and kidney uptake. Ultrastructure of Kupffer cells revealed PFCE inclusions and extensive vacuolization. These experiments demonstrate that the clearance kinetics and organ distribution of circulating P. aeruginosa and their subsequent killing are altered by PFCE. Diminished hepatic phagocyte function leads to a decrease in vascular clearance of circulating bacteria, increased uptake in other reticuloendothelial organs, and decreased bactericidal activity versus P. aeruginosa.Because mononuclear phagocytes take up perfluorochemical emulsions (PFCE), we examined how prior treatment with PFCE affects the fate of circulating bacteria. Rats were preinjected with three daily intravenous injections of PFCE (2.0 ml/100 g) containing 12.5% (vol/vol) of a 4:1 mixture of F-dimethyl adamantane and F-trimethylbicyclo-nonane, 2.5% (wt/vol) Pluronic F-68 as the emulsifying agent, and 3% (wt/vol) hydroxyethyl starch as the oncotic agent. Pseudomonas aeruginosa or Staphylococcus aureus were injected 4 h after the third PFCE injection. PFCE pretreatment decreased the rate and extent of vascular clearance of P. aeruginosa, with decreased uptake by the liver. Importantly, there were significant decreases in killing of P. aeruginosa in the liver, lungs, spleen, and kidneys of PFCE animals. PFCE did not alter the clearance of S. aureus from the circulation. However, hepatic uptake was reduced, with concomitant increases in lung and kidney uptake. Ultrastructure of Kupffer cells revealed PFCE inclusions and extensive vacuolization. These experiments demonstrate that the clearance kinetics and organ distribution of circulating P. aeruginosa and their subsequent killing are altered by PFCE. Diminished hepatic phagocyte function leads to a decrease in vascular clearance of circulating bacteria, increased uptake in other reticuloendothelial organs, and decreased bactericidal activity versus P. aeruginosa.

Angiocentric recruitment of lymphocytes into the lung after the intrabronchial instillation of antigen

The pathogenesis of acute lymphocytic inflammation in the lower respiratory tract appears to involve the recruitment of lymphocytes out of the blood stream and into the extravascular lung tissue. To investigate the membrane molecules regulating this process, we used the intrabronchial instillation of cellular antigen to trigger lymphocyte recruitment into the lower respiratory tract. Sheep presensitized 6 to 10 weeks earlier at a remote site were intrabronchially challenged with 1.5 X 107 cells from a B lymphoblastoid cell line. The cells were instilled into a subsegmental bronchus through a bronchial catheter. The stimulated and contralateral control segments were studied at a peak of inflammation, approximately 72 hours after antigen stimulation. Gross and microscopic studies of the stimulated segment demonstrated localized inflammation characterized by the perivascular infiltration of lymphocytes. In contrast, control areas of the lung demonstrated only scattered perivascular lymphocytes. Immunohistochemistry of the stimulated lung showed that the majority of these perivascular cells were CD3+ CD4+ lymphocytes. The T lymphocytes expressed high levels of the cell adhesion molecules beta1 integrin and LFA-1, but low levels of the L-selectin membrane molecule. Immunohistochemistry of the endothelial cells associated with the lymphocyte infiltrates demonstrated intense staining of the ICAM-1, and beta1 integrin adhesion molecules. Electron microscopic studies of the endothelial cells in the antigen stimulated areas of the lung confirmed morphologic changes consistent with endothelialitis. These results suggest that the intrabronchial instillation of cellular antigen stimulates an angiocentric T-cell infiltration regulated by activated pulmonary endothelial cells. The histologic and morphologic findings are remarkably similar to those observed during acute lung transplant rejection.