Rabbit Alveolar Macrophages Research Articles

Effect of volume and pH on surface receptor number in macrophages.

Incubation of rabbit alveolar macrophages in hypo-osmotic solutions transiently increases cell volume and inhibits membrane internalization, resulting in an increase in surface receptor number. Since recent reports suggest that hypo-osmotic treatment decreases intracellular pH, and that reduced pH inhibits receptor internalization, pH was measured in hypo-osmotically treated macrophages. We found that cells incubated in iso-osmotic solutions of pH less than 7.2 exhibited a decrease in intracellular pH upon exposure to hypo-osmotic solutions, while cells in iso-osmotic solutions of pH greater than 7.2 had an increase in pH upon exposure to hypo-osmotic solutions. The relative increase in surface receptor number was unaffected by the initial pH or by the direction of change in pH. Incubation of cells in high K+/low Na+ hypotonic buffers induced a persistent increase in cell volume and surface receptor number. Cell volume and surface receptor number fell to baseline values after restoration of isotonicity by the addition of hypertonic sucrose. These manipulations had little effect on intracellular pH. We conclude that the inhibition of membrane internalization observed in cells exposed to hypo-osmotic solutions is independent of changes in intracellular pH. The inhibition of internalization observed in this system may be due directly to forces produced as a consequence of cell swelling.

Eicosanoids produced during interactions between Pseudomonas aeruginosa and alveolar macrophages are species‐dependent

Eicosanoid production during phagocytosis of pyogenic bacteria by rabbit alveolar macrophages was studied as a model of early events in the pathogenesis of pneumonia. Adherent alveolar macrophages, prelabelled with [3H]-arachidonic acid (AA), were incubated with live, opsonized Staphylococcus aureus or Pseudomonas aeruginosa (bacteria:macrophage ratio of 50:1) at 37 degrees C for 90 min. Supernatant eicosanoids were extracted and separated by reverse phase high performance liquid chromatography (RP-HPLC). While the amounts of labelled PGE2, TXB2, and PGD2 produced in response to the two organisms were equal, the amount of PGF2 alpha elicited by S. aureus amounted to three times that released during macrophage challenge with P. aeruginosa. Overall, preferential release of cyclooxygenase products occurred during phagocytosis of S. aureus. In contrast, eicosanoids identified presumptively as oxygenated metabolites of AA predominated in cultures challenged with opsonized P. aeruginosa. Live, non-opsonized P. aeruginosa elicited the same profile of eicosanoids, but in reduced amounts. Inhibitor studies indicated that these AA derivatives were not synthesized via the macrophage lipoxygenase pathway. Their production was dependent on the viability of P. aeruginosa. Macrophages challenged with opsonized, heat-killed P. aeruginosa resulted in production of an eicosanoid profile similar to that elicited by S. aureus. Secondary metabolism by P. aeruginosa of eicosanoids released from the macrophage did not contribute to the unique profile produced during the interaction of this organism with labelled macrophages. Our data indicate that during binding to macrophages, the primary human pathogen, P. aeruginosa, specifically modulates the profile of eicosanoids produced. This effect on inflammatory mediators may be of biological significance in the pathogenesis of pneumonia.

Tannin mediated alveolar macrophage protein phosphorylation

Experiments were designed to determine if cotton bract tannin, a component of cotton mill dust, would promote the phosphorylation of alveolar macrophage proteins in doses potentially achievable in vivo. Rabbit alveolar macrophages were loaded with 32PO4 and challenged with various doses of tannin for time periods ranging from two seconds to 120 minutes. Changes in protein phosphorylation began after two seconds and were maximal at five to fifteen minutes. Dose response studies using an exposure time of one hour showed phosphorylation changes began at 1 micrograms/mL and were maximal at 10 to 30 micrograms/mL. Phosphorylation changes were similar to but not identical to those induced by the protein kinase C activator, phorbol myristate acetate (PMA). Calcium ionophore, A-23187 had no clear effect either alone or in conjunction with PMA. These results indicate that cotton bract tannin is able to rapidly promote protein phosphorylation of alveolar macrophages at doses potentially achievable in vivo. Other mechanisms in addition to those of protein kinase C appear to be involved in this protein phosphorylation process.

Toxicity of metallic ions and oxides to rabbit alveolar macrophages

The effects of soluble compounds and oxides of As, Cd, Cu, Hg, Ni, Pb, Sb, Sn, V, and Zn on oxidative metabolism and membrane integrity of rabbit alveolar macrophages were studied by 24-hr in vitro exposure. Oxidative metabolism induced by phagocytosis of opsonized zymosan was measured by H2O2 and O2- release and by chemiluminescence in the presence of luminol. Membrane integrity was estimated by extracellular LDH activity. Metallic ions and oxides inhibited the release of active oxygen species. Cd(II), As(III), and V(V) were the most toxic elements as measured by all investigated parameters. Cu(II) decreased O2- release and chemiluminescence effectively but H2O2 release and membrane integrity less. Chemiluminescence was decreased strongly by Hg(II) while O2- and H2O2 release were depressed moderately. Zn(II) and Sb(III) compounds caused medium toxicity and the tested Sn, Ni, and Pb compounds showed only faint toxic effects.

Serum myoglobin occurs mainly in the form of circulating immunocomplexes

The penetration of FITC-fibrinogen in rabbit alveolar macrophages depends on Ca2+ and Mg2+. Therefore there is reason to believe the process is not restricted only by diffusion on concentration gradient, but pinocytosis takes place, which depends Ca2+. The absorption of FITC-label from fibrin by alveolar macrophages is more slow than from fibrinogen. Soluble high-molecular products of fibrin degradation can be trapped by pinocytosis. On the basis of such studies it was assumed that degradation of fibrin proceeds parietally.

L-Fucose blocks MIF/MAF priming of rabbit alveolar macrophages for a PMA-induced oxidative response

The effect of l-fucose on the priming of AM from normal adult rabbits and their subsequent chemiluminescent (CL) responses to phorbol myristate acetate (PMA) was investigated. It was observed that 12.5 m M l-fucose, but not d-fucose, blocked the “spontaneous” priming of normal AM during an 18-hr incubation period in serum-free RPMI 1640 medium by about 40% ( P < 0.01) as detected by their CL responses to a PMA challenge. In addition, the optimal concentration of l-fucose (12.5 m M) blocked MIF/MAF priming during the 3- or 18-hr incubaion period by 71 or 93% ( P < 0.05), respectively, as determined by their CL responses following PMA challenge. It is of particular significance that d-fucose was inactive. These results, together with previously published data, indicate that l-fucose (a) blocks priming of AM for an oxidative response, (b) stimulates random migration of AM, and (c) reverses migration inhibition produced by migration inhibition factor.

家兎の活性化肺胞マクロファージを認識する単クローン性抗体, AM-1の検討

家兎の活性化肺胞マクロファージを認識する単クローン性抗体, AM-1の検討

Intraphagosomal pH in Alveolar Macrophages Studied with Fluorescein-Labeled Amorphous Silica Particles

Intraphagosomal pH in rabbit alveolar macrophages was studied using amorphous silica particles (FSP) and yeast particles (FYP) labeled with fluorescein. The pH was estimated from the quotient between the fluorescence intensity at wavelength 519 nm with excitation at wavelengths 495 and 450 nm. Within a factor of 10, pH was independent of the number of FSP added to macrophages in vitro. In macrophages cultured for 24 h, the pH obtained with FSP and FYP was about 5. Three hours after lavage, pH was the same as after 24 h for the FSP but significantly higher for the FYP, 5.8. Both 3 and 24 h after lavage, more lysosomes were in contact with the FYP-containing phagosomes than with the FSP-containing ones. Most FSP were in tight contact with the phagosomal membrane, while there was a clear zone between most FYP and the phagosomal membrane. The differences in pH and morphology between cells containing FSP and FYP might be explained by the assumptions that the macrophages disintegrate the FYP, which results in higher pH, and that the disintegration of FYP is more efficient at 3 than at 24 h after the lavage. The intraphagosomal pH was lower when the macrophages were allowed to phagocytize the FSP in vivo. The pH values were 5.1 in vitro, 4.9 at 24 h, and 4.5 at 1 week after the FSP had been instilled via trachea. The FSP should be a useful tool for estimation of intraphagosomal pH at basic conditions, i.e., the milieu to which many inhaled inorganic particles will be exposed.

Effects of Extracellular Slime Produced by Staphylococcus epidermidis on Oxidative Responses of Rabbit Alveolar Macrophages

Bacterial slime produced in mass cultures of the RP 12 strain of Staphylococcus epidermidis was extracted with 4 M guanidine-HCl plus 0.05 M sodium acetate and 0.5% CHAPS, concentrated, dialyzed, and subjected to separation on DEAE sephacel columns. Three fractions, I-2A, I-2B, and I-4, were eluted with linear gradients of NaCl. Fractions I-2A and I-2B were alcian blue positive, whereas I-4 was alcian blue negative but the most electronegative fraction. The crude polysaccharide fraction and the three purified fractions were incubated individually for 2.5 or 20 h with normal rabbit alveolar macrophages (AM) to determine their effect on a subsequent PMA-induced oxidative burst. The crude fraction (50-200 micrograms/mL) and I-2B (50-200 micrograms/mL) primed the AM to give approximately a threefold increase in the PMA-induced burst after 2.5 h incubation. In contrast, a 20-h incubation resulted in a 30-40% inhibition of the PMA-induced burst with AM incubated with the same concentrations of the crude, I-2A, or I-2B fractions. Fraction I-4 had no detectable effect. The fractions also were tested to determine if they could elicit an oxidative burst in BCG-immune AM. None of the fractions (up to 500 micrograms/mL) elicited a significant oxidative burst even though BCG-immune AM yielded a PMA-induced burst 100 times that observed with normal resident AM. These data suggest that slime from S. epidermidis can impair the PMA-induced oxidative burst of normal AM during a 20-h incubation period and could explain in part why host defenses are compromised by slime-producing S. epidermidis.

Tannin-mediated Secretion of a Neutrophil Chemotactic Factor from Alveolar Macrophages: Potential Contribution to the Acute Pulmonary Inflammatory Reaction Associated with Byssinosis

Inhalation of cotton mill dust or condensed tannin, a major botanical component of cotton mill dust, induces an acute pulmonary inflammatory response characterized by a rapid influx of neutrophils into the airways. The development of neutrophil alveolitis caused by tannin inhalation is accompanied by the accumulation of low molecular weight neutrophil chemotactic factor (NCF) in the airways. To determine if the alveolar macrophage is the source of this NCF, the ability of tannin to induce the secretion of NCF from rabbit alveolar macrophages was examined in vitro. Tannin did promote the secretion of NCF from alveolar macrophages in a time- and dose-dependent manner. Secretion began immediately after challenge and was maximal after 4 to 5 h. Maximal secretion occurred at a tannin concentration of 50 micrograms/ml. Comparison with the dose response for NCF secretion by cotton dust extract indicated that tannin was the major component in the dust responsible for NCF secretion from alveolar macrophages in the time period examined. The NCF had an apparent molecular weight of greater than 800 as determined by gel chromatography. The NCF could be extracted into organic solvents, suggesting it was a lipid. Its secretion, however, could not be prevented by treatment of the macrophages with the 5-lipoxygenase inhibitor, nordihydroguaretic acid, demonstrating that the NCF was not leukotriene B4. These data indicate that the action of tannin on resident alveolar macrophages results in the secretion of a NCF that may be responsible for the acute neutrophil alveolitis associated with inhalation of cotton dust.

The Effect of Cadmium on Arachidonate Release in Rabbit Alveolar Macrophages (Proceedings of the 14th Symposium on Environmental Pollutants and Toxicology)

The Effect of Cadmium on Arachidonate Release in Rabbit Alveolar Macrophages (Proceedings of the 14th Symposium on Environmental Pollutants and Toxicology)

Estimation of pH in individual alveolar macrophage phagolysosomes.

A method for measurement of phagolysosomal pH in individual alveolar macrophages based on a cytofluorometric technique with fluorescein-labeled silica particles (FSP) as a probe was developed. The size of the FSP, 3.0 or 5.0 microns, did not affect the result of the pH measurements. The average pH values, range 5.1-5.5, of individual particles in macrophages from three rabbits agreed well with the pH values obtained in a macrophage population using a fluorescence spectrometer and the FSP. The variation of pH in phagolysosomes in alveolar macrophages from five rabbits was investigated. Measurements were performed 3, 6, and 24 h after addition of the FSP in cells containing one particle as well as in cells containing two particles. The variation in pH was small, with a coefficient of variation less than 10% in all rabbits at all times. There was a significant correlation between values obtained from two phagolysosomes in the same cell, indicating a cell factor responsible for 10-30% of the total variance. The fact that the variation of pH is small in normal, untreated rabbit alveolar macrophages should be of importance when estimating alveolar clearance of inorganic particles due to dissolution in the acid phagolysosomal milieu.

Isolation of low density lipoprotein from atherosclerotic vascular tissue of Watanabe heritable hyperlipidemic rabbits.

Atherogenic properties of low density lipoproteins (LDL) in vivo may reflect modification of lipoproteins associated with endothelial translocation and exposure to extracellular matrix and interstitial fluid. To examine whether modifications of LDL occur in vivo, lipoproteins were isolated from plasma and vascular tissue of Watanabe heritable hyperlipidemic (WHHL) rabbits. LDL was extracted from vascular tissue (LDL-V) by homogenization in the presence of a sodium carbonate buffer. Control experiments demonstrated that modifications did not occur under the preparative conditions used to release LDL from tissue. LDL-V contained less esterified cholesterol, but more cholesterol esters, than did LDL from plasma (LDL-P). The diameters of both LDL-V and LDL-P followed gaussian distributions, but LDL-V particles were smaller (20.3 +/- 0.1 and 26.3 +/- 0.1 nm). Mild lipid peroxidation was evident in LDL-V. The sphingomyelin content was increased in LDL-V, with less phosphatidylcholine than in LDL-P. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that apolipoprotein B was depleted in LDL-V, but Western blot analyses identified lower molecular weight proteins antigenically related to apolipoprotein B. LDL-V markedly stimulated cholesterol esterification in mouse peritoneal macrophages and also in rabbit alveolar macrophages, a cell type that did not respond to acetylated LDL. LDL-V was not recognized by cultured rabbit skin fibroblasts. Thus, LDL isolated from atherosclerotic vascular tissue in vivo was modified in a fashion that could confer atherogenic properties reflected by augmentation of cholesterol esterification in macrophages in vitro.

Selective inhibition of free arachidonic acid production in activated alveolar macrophages by calmodulin antagonists

The effect of calmodulin antagonists on the amounts of free fatty acids produced by rabbit alveolar macrophages was determined by fluorometric high-performance liquid chromatography. Opsonized zymosan-induced arachidonic acid production was dramatically suppressed in the presence of W-7 and trifluoperazine without an effect on the production of other fatty acids. Calmodulin antagonists inhibited phospholipase A and abolished the release of arachidonic acid from phospholipids. The present results suggest that a zymosan-sensitive pool of 20:4, which is different from that of other fatty acids, is present in macrophages and that calmodulin antagonists selectively inhibit phospholipase A, which preferentially degrades phospholipids with 20:4.

Concurrent expression of procoagulant and plasminogen activator activities by rabbit alveolar macrophages in vitro: Opposite modulating effects of prostaglandin E 2

We examined the effects of arachidonic acid metabolites on the simultaneous expression of procoagulant (PC) and plasminogen activator (PA) activities by rabbit alveolar macrophages. Incubation with lymphocyte-conditioned medium (LCM) caused a significant increase in cell-associated PC activity. Co-treatment with indomethacin (1 μM) reduced this augmentation in PC activity by 33% (p < 0.05). In contrast, indomethacin caused a 42% increase in PA activity released into incubation medium (p < .05). Both effects of indomethacin were reversed by the addition of PGE 2 in concentrations as low as 1 nM. Addition of 100 nM PGE 2 to these cells caused an increase in PC activity 2.7-fold greater than that achieved by LCM alone, while PGE 2 suppressed released PA activity by 62%. PGE 2 and indomethacin had similar but less pronounced effects on phorbol myristate acetate-treated cells. These effects of PGE 2 could be duplicated by PGE 1, but not by any other arachidonic acid metabolite (PGF 2α, PGI 2, PGD 2, ddPGF 2α, LTB 4, or LTC 4). While PGE 2 increases intracellular levels of cAMP, the observed effects on PC and PA activities could not be reproduced fully by treatment with dibutyryl cAMP. We conclude that PGE 2 amplifies the augmentation of PC activity by stimulated alveolar macrophages while concurrently inhibiting expression of plasminogen activator. This suggests that PGE 2 may be a significant mediator in regulating the highly interactive processes of inflammation and coagulation/fibrinolysis.

Alveolar macrophages in rabbits after combined exposure to nickel and trivalent chromium

Rabbits were exposed to a combination of 0.7 mg/m3 Ni2+ as NiCl2 and 1.2 mg/m3 of Cr3+ as Cr(NO3)3, to 0.6 mg/m3 of Ni2+ as NiCl2, or to filtered air for about 4 months, 5 days/week and 6 hr/day. Alveolar macrophages were recovered by lung lavage and studied by light and electron microscopy. Metabolic activity, phagocytic capacity and lysozyme activity in the macrophages were studied. After the combined exposure, the effects on lung weight, number of macrophages, and appearance of surface and number of intracellular laminated inclusions in these cells were more than additive. These effects might be explained by a combination of increased production by Ni2+ and impaired catabolism of surfactant by Cr3+. Because the metal concentrations used were not far above occupational threshold limit values, combined exposures to nickel and trivalent chromium should be considered more seriously.

Discussion

Discussion

Secretion of the lysosomal acid triacylglycerol hydrolase precursor by J774 Macrophages

J774, thioglycollate-elicited mouse peritoneal and BCG-induced rabbit alveolar macrophages all contain high levels of a triacylglycerol hydrolase (EC 3.1.1.3) (TGase) with optimal activity at pH 6.5. The J774 macrophages, a cell line deficient in the calcium-independent mannose 6-phosphate receptor, were found to secrete large quantities of the TGase into the culture medium. In contrast, mouse peritoneal and rabbit alveolar macrophages, which are both mannose 6-phosphate receptor-competent cell types, secreted much lower amounts of neutral TGase. The enzyme was localized in the lysosomes of rabbit alveolar macrophages. Addition of 25 mM NH 4Cl induced a 6-fold increase in TGase secretion by alveolar macrophages, while 50 mM NH 4C1 induced a 12-fold increase in TGase secretion. NH 4Cl had no effect on TGase secretion by J774 macrophages. The TGase secreted by J774 macrophages was internalized by I-cell disease fibroblasts, increasing the cellular content of TGase 10-fold after 8 h. Internalization was inhibited 70% by the addition of 2 mM mannose 6-phosphate to the culture medium, but was not affected by 2 mM mannose or glucose 6-phosphate. After internalization, the neutral TGase was converted to a TGase with a pH optimum of 5.1. These data are consistent with the spontaneous release of a lysosomal enzyme precursor from a calcium-in-dependent mannose 6-phosphate receptor-deficient cell line, indicating that the neutral TGase previously reported in several types of macrophages may be the precursor of the lysosomal acid TGase.

Cathepsin D is membrane-associated in macrophage endosomes.

Previously we identified an acid protease activity which was located in the endosomes of rabbit alveolar macrophages (Diment, S., and Stahl, P.D. (1985) J. Biol. Chem. 260, 15311-15317). In this study, the endosomal protease is identified as cathepsin D by immunoprecipitation with polyclonal antibodies raised against rabbit cathepsin D and by NH2-terminal sequence. In order to elucidate the mechanism for targeting of cathepsin D to endosomes, we first examined the membrane association of cathepsin D with light (rho = 1.05 g/ml) and heavy density (rho = 1.1 g/ml) vesicles from Percoll density gradients. After sequential washes, 8.4 and 21.9% of cathepsin D activity remained associated with heavy and light density vesicles, respectively. This membrane-associated cathepsin D could not be solubilized in either buffer at pH 5.0 containing mannose 6-phosphate and EDTA or in buffer at pH 10.6. Solubilization required the detergent Triton X-100. To determine whether membrane-associated cathepsin D was found in endosomes, the enzyme was radioiodinated within endosomes and lysosomes with internalized lactoperoxidase. The membrane-associated form was detected in endosomes, but much less in lysosomes. Biosynthetic studies combined with the same extraction procedure revealed that macrophage cathepsin D is first synthesized as an inactive membrane-associated precursor. The precursor is processed to an active, membrane-associated form and then to the active soluble form found in lysosomes. Our studies provide evidence that 1) cathepsin D is in endosomes of macrophages; 2) cathepsin D is transported to endosomes as a membrane-associated form; and 3) the membrane-associated form is a biosynthetic precursor for the soluble form found in endosomes and lysosomes.

Alveolar macrophage-stimulated neutrophil and monocyte migration: Effects of in vitro ozone exposure

The ability of rabbit alveolar macrophages (AM) to release factors which stimulate the migration of peripheral blood neutrophils and monocytes was examined, and the influence of in vitro ozone exposure on this secretory activity was investigated. To evaluate the ability of AM to release leukocyte chemotactic activity, AM obtained by bronchoalveolar lavage were established in monolayer or suspension culture, with and without added zymosan, for 2 and 6 hr. The resulting macrophage-conditioned medium was tested for chemotactic activity using modified Boyden-type chambers and rabbit peripheral blood neutrophils or monocytes as the responding cells. The results demonstrate that substrate attachment (monolayer culture) and/or zymosan phagocytosis can stimulate AM to release chemoattractants for monocytes and neutrophils. Additionally, the results suggest that AM are constitutively producing low levels of monocyte chemotactic factors. The effects of in vitro ozone exposure on the secretion of chemotactic activity was investigated by exposing monolayer cultures of AM to air, 0.1, 0.3, or 1.2 ppm ozone for 2 hr. Macrophage-conditioned medium was harvested immediately, 2 and 6 hr postexposure, and tested for chemotactic activity. Exposure to 0.3 and 1.2 ppm ozone significantly increased the AM secretion of factors which stimulated neutrophil migration; additionally, the results strongly suggest that ozone can augment the ability of AM to stimulate monocyte migration. These results imply a role for the AM in the recruitment of inflammatory cells after ozone inhalation.