Study of rat pulmonary macrophages as affected by hydrocortisone administration and adrenalectomy

The blood clearance rate of inert colloidal particles and the number of rat lung interstitial phagocytic cells decrease considerably on the 7th day after daily subcutaneous hydrocortisone acetate (HC) injection at a dose of 125 mg/kg. The number of cells in the bronchoalveolar lavage (BAL) increases more than 5-fold, and the absolute quantity of neutrophils is 66 times higher than in the control. Phagocytic and microbicidal activity of HC-treated animal alveolar macrophages (AM) decreases. Stimulation with zymosan led only to the recovery of the normal parameters of mononuclear phagocytosis system (MPC) and its pulmonary compartment activity. The parameters of MPS and AM studied increase on the 7th day after bilateral adrenalectomy (AE). The number of BAL cell increases 1.4-fold due to the animals' death immediately after intravenous zymosan injection because of total hemorrhage. The data obtained testify to the influence of glucocorticoids on the composition and activity of bronchoalveolar space cells, which in turn determine the resistance of the lung tissue.     

Lipid peroxidation in the rat liver during macrophage stimulation

The content of malonyl dialdehyde, one of the final products of lipid peroxidation, was determined at different stages after zymosan stimulation of mononuclear phagocyte system in the rat liver. The gradual increase (with the maximum on day 7) of colloidal carbon clearance from the blood and the number of nonparenchymal cells, especially their nonphagocytic fraction, was noted in the rat liver after zymosan injection. A considerable reduction of hepatic malonyl dialdehyde was observed after the initial (on day 1) rising of its level. The distinct antioxidant effect developed by day 7 and coincided with maximal activity of hepatic macrophages.     

Change in the absorptive capacity of macrophages from different organs after administration of hydrocortisone

Two, twenty-four and 48 h after hydrocortisone treatment in a dose of 125 mg/kg bw the blood clearance rate for colloidal carbon particles in rats turned to be 2, 2.1. and 1.6 times less whereas that for 51Cr-SRBC in CBA mice 2.1, 2.2 and 1.7 times less as compared to untreated controls. Within 24 and 72 h after hormone injection the efficacy of red blood cell uptake by Kupffer cells decreased 1.35 and 1.8 times whereas the similar uptake by lung or spleen macrophages changed but insignificantly and that by bone marrow cells was even greater than in controls. Toward the 5th day after zymosan treatment the uptake capacity of Kupffer cells was the greatest whereas the plasma 11-OHCS content was 1.3-fold less versus the control values.     

Endocytosis by liver cells during stimulation of the mononuclear phagocyte system by yeast polysaccharides]

Endocytosis (phagocytosis, fluid-phase- and receptor-mediated endocytosis) by liver cells, lysosomal enzyme activities have been studied during macrophages stimulation by yeast polysaccharides. It was shown that like macrophages stimulator zymosan, yeast polysaccharides cryelan and rhodexman increased the carbon particles phagocytosis. The most effective was intravenous administration of yeast polysaccharides. Compared to rhodexman, the effect of cryelan was more prominent. Macrophages stimulation was followed by suppression of fluid-phase endocytosis by liver cells. Increased activity of cathepsin B was discovered on day 5 after macrophages stimulation (proteinase, most typical for macrophages enzymes).     

Dependence of mononuclear infiltration of the liver on hepatocyte proliferation

Partial liver resection (two-thirds) performed in (CBA X C57BL)F1 mice 2-4 or 24 h after intravenous injection of 2 mg zymozan led to retardation of the formation of mononuclear infiltrates in the liver and the lung. In control sham-operated animals, the first signs of infiltration appeared on the 2nd, whereas in mice with partial liver resection ( PLR ) on the 5th day after zymozan -induced stimulation of liver macrophages. If mice underwent PLR 5 days after zymozan injection, the preformed mononuclear infiltrates experienced the reverse development. PLR did not abolish monocytosis which was observed after zymozan injection. On the other hand, when mice received hydrocortisone in a dose of 125 mg/kg, the zymozan -induced infiltration in the liver as well as monocytosis were blocked. It is assumed that depression of mononuclear infiltration in the liver and the lung after liver resection is linked with a specific effect of proliferating hepatocytes rather than with the stress-induced mobilization of glucocorticoids.     

Functional restructurings of the mononuclear phagocyte system in experimental liver cirrhosis

In rats with CCl4-induced liver cirrhosis the clearance rate of colloid carbon particles was more than 2 times lower than in control animals. Simultaneously the uptake capacity of liver Kupffer calls falls. The number of phagocytizing liver macrophages decreased. Along with the diminished functional activity of liver macrophages in cirrhotic liver, the total number of lung and spleen macrophages increased 1.5-fold, with their uptake capacity increasing 10- and 3-fold, respectively. The nitroblue tetrazolium dye reduction and methacrylate particles uptake by alveolar macrophages in vitro rises. The liver, lung, spleen and peritoneal macrophages during liver fibrosis become less sensitive to zymosan stimulation. The incidence of zymosan-induced liver infiltrates decreases 50-fold, while in the lungs they do not develop at all. Such a decreased macrophage reactivity may be closely linked with progressing, poorly reversible liver fibrosis.     

Corticosterone reception by alveolar macrophages when their functional activity has changed

The binding of 3H-corticosterone by rat alveolar macrophages was studied before and after stimulation with zymosan in vivo. Thirty min after incubation of the macrophagal monolayer from intact animals with 3H-corticosterone accumulation of the hormone by the cells came to an end. As the concentration of 3H-corticosterone in the incubation medium was raised, the binding of the hormone with the saturated (receptor) system of alveolar macrophages terminated upon absorption of 10.6 fmol per 10(6) cells. Further raising of the level of the bound hormone was effected by the unsaturated (lipid) system. Stimulation with zymosan led not only to an increase in the number of the cells of the bronchoalveolar tract but also to an elevation of the intensity of 3H-corticosterone engulfment by alveolar macrophages. The number of binding sites per cell in the zymosan-activated macrophages increased 1.5-fold. This may be an important moment determining the development and liquidation of mononuclear infiltrations in the lung.     

The development of lysosomal apparatus. II. Incorporation,subcellular distribution,and intraparticulate hydrolysis of 131 I-albumin by liver of mice at perinatal stages

Mouse fetuses at 15th and 18th day of gestation, and newborns aging 0, 5, 10, and 15 days were injected i.p. with 131 I-albumin (RISA). The degree of incorporation by liver and the intraparticulate hydrolysis of RISA in 27,000g × 10 min. particles increased after birth. By this time, changes in subcellular distribution of RISA and marker enzymes were also observed. Following an i.p. injection of India ink, numerous hepatic cells stained with carbon particles were observed at the light microscope from day 5 of life. The results suggest that the lysosomal apparatus acquires full capacity to incorporate colloidal particles and to degrade foreign macromolecules in the first week of life.     

Biochemical differences in the mechanism of macrophage lysosomal exocytosis initiated by zymosan particles and weak bases.

By utilizing compounds with different inhibitory properties, discrete biochemical differences were found in the mechanism of selective lysosomal enzyme secretion by macrophages in response to stimulation with zymosan particles and methylamine. Pretreatment of macrophages with trypsin markedly impaired the capacity of the cells to respond to stimulation with zymosan particles, but had no effect on methylamine-stimulated lysosomal enzyme secretion. Similarly, the addition of phenylmethanesulphonyl fluoride or EDTA to the incubation medium substantially inhibited zymosan-induced lysosomal enzyme secretion, whereas the methylamine-stimulated response was unaffected by these agents. The addition of 2-deoxyglucose to incubation media, however, strongly inhibited both zymosan- and methylamine-stimulated beta-galactosidase secretion. These findings are consistent with a mechanism for lysosomal enzyme secretion by macrophages, based on a receptor-dependent uptake of zymosan particles and a receptor-independent uptake of methylamine.     

Plasma membrane phospholipid translocation in the mouse peritoneal macrophage: differential response to stimulation of eicosanoid production.

The metabolism and translocation of exogenously introduced plasma membrane phosphatidylcholine (PC) having the fluorescent fatty acid analog aminocaproyl NBD (N-nitrobenzo-2-oxa-1,3 diazole) (NBD-PC), in the sn2 position was studied in cultured murine peritoneal macrophages using biochemical and morphological techniques. Following labeling of the cell plasma membrane at 2 degrees C by vesicle lipid exchange, macrophages were warmed in the presence or absence of pharmacological stimuli of eicosanoid production and release. Fluorescence microscopy indicated that the phospholipid was translocated to an internal cellular pool upon stimulation with zymosan. In contrast, the membrane PC analog was primarily metabolized and released after being found diffusely associated with the cytoplasm in macrophages stimulated with the calcium ionophore A23187. Evidence obtained by double labeling zymosan-treated macrophages with NBD-PC and a monoclonal antibody directed against a lysosomal membrane protein demonstrated that the fluorescent lipid is internalized in association with the zymosan particles and both are found in lysosomes. The results suggest that multiple pathways exist in peritoneal macrophages which target plasma membrane PC into different cellular compartments for hydrolysis and conversion to eicosanoid products and release from cells.     

Relationship of prostaglandin secretion by rabbit alveolar macrophages to phagocytosis and lysosomal enzyme release

The phospholipids of rabbit alveolar macrophages were pulse-labelled with [(14)C]-arachidonic acid, and the subsequent release of labelled prostaglandins was measured. Resting macrophages released measurable amounts of arachidonic acid, the prostaglandins E(2), D(2) and F(2alpha) and 6-oxoprostaglandin F(1alpha). Phagocytosis of zymosan increased the release of arachidonic acid and prostaglandins to 2.5 times the control value. In contrast, phagocytosis of inert latex particles had no effect on prostaglandin release. Indomethacin inhibited the release of prostaglandin, and, at high doses (20mug/ml), increased arachidonic acid release. Analysis of the cellular lipids showed that after zymosan stimulation the proportion of label was decreased in phosphatidylcholine, but not in other phospholipids or neutral lipids. Cytochalasin B, at a dose of 2mug/ml, inhibited the phagocytosis induced by zymosan but increased prostaglandin synthesis to 3.4 times the control. These data suggest that the stimulation of prostaglandin synthesis by zymosan is not dependent on phagocytosis. Exposure to zymosan also resulted in the release of the lysosomal enzyme, acid phosphatase. Furthermore, cytochalasin B augmented the zymosan-stimulated release of acid phosphatase at the same dose that stimulated prostaglandin synthesis. However, indomethacin, at a dose that completely inhibited prostaglandin synthesis, failed to block the lysosomal enzyme release. Thus despite some parallels between the release of prostaglandins and lysosomal enzymes, endogenous prostaglandins do not appear to mediate the release of lysosomal enzymes. The prostaglandins released from the macrophages may function as humoral substances affecting other cells.     

Corticosteroids and lung surfactant levels in adult male rats

Following lung instillation in adult male rats of 3.4 mumol hexavalent chromium (K2Cr2O7) dissolved in 0.5 ml of 0.9% NaCl, increased levels of lung surfactant could be detected after 48 h. The blood serum concentration of corticosterone was elevated in these animals. Blood serum thyroxine and triiodothyronine showed an initial increase after lung instillation of hexavalent chromium followed by a decline. Metabolism of testosterone by the alveolar macrophages to 17 beta-hydroxy-5 alpha-androstane-3-one and 5 alpha-androstane-3 alpha, 17 beta-diol was reduced 6 and 12 h after the K2Cr2O7 instillation, which was also associated with damage of lung cell function and decreased uptake by the alveolar macrophages of Candida albicans particles. As early as 12 h after s.c. administration of 400 micrograms dexamethasone/100 g body wt, increased levels of lung surfactant could be measured. At this time the lungs showed no signs of cellular damage, and metabolism of testosterone as well as uptake of Candida albicans particles by the alveolar macrophages were normal. Lower s.c. doses of dexamethasone did not result in raising the levels of lung surfactant in 12 h. Within 12 h after s.c. administration of large doses of testosterone, dihydrotestosterone or dehydroepiandrosterone no measurable effects on the levels of lung surfactant could be measured. Since animals treated with dexamethasone (200 micrograms/100 g body wt) or long-acting synthetic ACTH (100 micrograms i.m. Synacthen Depot/100 g body wt) for 5 days after lung instillation of K2Cr2O7 had extremely high levels of lung surfactant, it is concluded that the corticosteroids in adult rats may help to create augmented surfactant levels following lung intoxication. This could proceed via stimulation of surfactant production and reduction of surfactant removal. Different aspects of lung surfactant metabolism are discussed.     

Prostaglandin synthesis and release by subpopulations of rat alveolar macrophages

Alveolar macrophages are the primary phagocytic cell of lung, but are also capable of a variety of other functions, which include initiating or modulating inflammatory and immune responses through the production of soluble mediators. One such group of mediators is the eicosanoids. Further, recent data indicate that alveolar macrophages are not functionally homogeneous, but are heterogeneous with several subpopulations that differ both morphologically and functionally. Considering the apparent importance of prostaglandin synthesis and release in inflammatory and immune responses, the current study was undertaken to determine whether alveolar macrophage subpopulations differ in their ability to synthesize and release prostaglandin (PG) E, PGI2, and thromboxane A2 after stimulation by calcium ionophore A23187, zymosan, or aggregated IgG. Alveolar macrophages were harvested by bronchoalveolar lavage and were separated into 18 density-defined fractions. Density-defined alveolar macrophages (DD-AM) showed marked heterogeneity in prostaglandin synthesis and release. Maximal PGE synthesis and release was seen as a single peak after calcium ionophore A23187 and zymosan stimulation. In contrast, two peaks in PGE synthesis were seen after aggregated IgG stimulation. PGI2 synthesis was seen as a single peak generated by different DD-AM after calcium ionophore A23187 and zymosan. In contrast, aggregated IgG stimulation of subpopulations exhibited uniform synthesis and release of PGI2. Thromboxane A2 synthesis and release was maximal from a broad range of various DD-AM after calcium ionophore A23187, zymosan, and aggregated IgG stimulation. The results demonstrate that DD-AM are heterogeneous in ability to synthesize and release prostaglandins which is dependent on the stimuli. Therefore, specific subpopulations of alveolar macrophages may be central to the control of the pulmonary inflammatory response through specific eicosanoid synthesis and release.     

Early ontogeny of monocytes and macrophages in the pig

Prenatal development of cord blood monocytes and tissue macrophages was studied in pig foetuses by immunophenotyping and functional assays. The function of peripheral blood monocytes was compared in germ-free and conventional piglets. First macrophages were identified by electron microscopy in foetal liver on the 25th day of gestation. Monoclonal antibodies against porcine CD45 and SWC3 antigens were used for flow cytometric identification of myelomonocytic cells in cell suspensions prepared from the yolk sac, foetal liver, spleen and cord blood. Leukocytes expressing the common myelomonocytic antigen SWC3 were found in all organs studied since the earliest stages of development. Opsonized zymosan ingestion assay was used to determine the phagocytic capacity of foetal mononuclear phagocytes isolated from cord blood, liver and spleen. In the foetal liver, avid phagocytosis of apoptic cells had been found to occur before cells were able to ingest zymosan in vitro. The first cells capable of ingesting zymosan particles were found on the 40th day of gestation in umbilical blood and 17 days later in foetal spleen and liver. Their relative proportion increased with age. Cord blood monocytes and peripheral blood monocytes in germ-free piglets had low oxidatory burst activity as shown by iodonitrophenyl tetrazolium reduction assay. A remarkable increase of oxidatory burst activity was observed in conventional piglets, probably due to activation of immune mechanisms by the microflora colonizing gastrointestinal tract.     

Relations between the changes of the phagocytic activity of the reticuloendothelial system and the alterations in protein and nucleic acids contents of the rat liver (author's transl)]

The phagocytic activity of the reticuloendothelial system (RES) was evaluated after a single oral administration of soya-bean oil to male rats (15 g/kg). 1. An emulsion of soya-bean oil administered to the rat by gastric intubation activated the phagocytosis of colloidal carbon; the stimulation appeared on the 2nd day after treatment and persisted up to the 3rd day. There was also a relation between the stimulation of RES activity, under our experimental conditions, and the increased level observed in the protein and nucleic acid contents of the liver. 2. In contrast, without emulsion, soya-bean oil depressed the phagocytic activity on the 2nd and 3rd days after administration of the oil. These changes were associated with a diminution in ribonucleic acid and protein contents of the liver. Although the mechanisms of soya-bean oil-induced alterations of phagocytic activity were not clarified, there was a relationship between the RES stimulation or inhibition and the modifications in nucleic acid and protein contents of rat liver.     

Interstitial macrophage subpopulations: responsiveness to chemotactic stimuli

Recent data suggest that interstitial macrophages are a heterogeneous group of cells with several subpopulations. This study was undertaken to determine if there is heterogeneity among rat interstitial macrophage subpopulations ability to respond to chemotactic stimuli. Alveolar macrophages were harvested and separated into density-defined fractions by centrifugation through a continuous iso-osmotic gradient of colloidal silica. Unfractionated and density-defined interstitial macrophages were then characterized as to their ability to migrate towards F-Met-Leu Phen and zymosan activated serum. Interstitial macrophages of density 1.053 and 1.083-1.097 gm/ml were found to have the greatest migrational movement towards F-Met-Leu-Phen which was lower than the unfractionated population. Interstitial macrophage subpopulations migrational movement towards zymosan activated serum exhibited a major peak by macrophages of density 1.053 gm/ml and a minor peak by macrophages of density 1.083-1.097 gm/ml which was lower than the unfractionated population. These results demonstrated that interstitial macrophages are heterogeneous in their migrational ability towards the chemotactic stimuli F-Met-Leu-Phen and zymosan activated serum and that there may be a cooperative interaction between the subpopulations which affects macrophages migrational ability.     

Regulation of macrophage eicosanoid production by hydroperoxy-and hydroxy-eicosatetraenoic acids.

Resident mouse peritoneal macrophages when exposed to zymosan during the first day of cell culture synthesize and secrete large amounts of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4), the respective products of cyclo-oxygenase- and 5-lipoxygenase-catalysed oxygenations of arachidonic acid. Under these conditions of cell stimulation only small amounts of hydroxyeicosatetraenoic acids (HETEs) are concomitantly produced. However, exogenously added arachidonic acid is metabolized to large amounts of 12- and 15-HETE and only relatively small amounts of PGE2. No LTC4 is formed under these conditions. In contrast, resident mouse peritoneal macrophages in cell culture for 4 days synthesized less PGE2 and LTC4 when exposed to zymosan. However, these macrophage populations continue to synthesize 12-HETE from exogenously added arachidonic acid. Zymosan induced the secretion of a lysosomal enzyme, N-acetyl-beta-glucosaminidase, equally in both 1- and 4-day cultures. Both 12- and 15-hydroperoxyeicosatetraenoic acids (HPETEs), the precursors of 12- and 15-HETE, were found to be irreversible inhibitors of the cyclo-oxygenase pathway and reversible inhibitors of the 5-lipoxygenase pathway in macrophages. 15-HETE were found to be reversible inhibitors of both pathways. Thus the oxidation of arachidonic oxidation of arachidonic acid to both prostaglandins and leukotrienes may be under intracellular regulation by products of 12- and 15-lipoxygenases.     

Behavior of magnetic particles of metallic iron in animals

Intraperitoneal introduction of 5-8 mu ferromagnetic iron particles into mouse leads to their capsulation in the liver and preservation in the organism during no less than 1 month. We have observed activation of peroxide lipid oxidation during 3-4 days after the introduction of particles and a two-fold increase of the free iron content in the liver. Intraperitoneal introduction of ferromagnetic particles with diameter below 1 mu produced rapid death. Intravenous injection of 5-8 mu diameter ferromagnetic particles into rat resulted in their accumulation in the liver, lung and spleen, probably due to the absorption of the iron particles by macrophages. In two weeks these particles were transformed and vanished from the tissues.     

Different patterns of arachidonate metabolism in autologous human blood monocytes and alveolar macrophages

As peripheral blood monocytes (PBM) differentiate into tissue macrophages, they undergo a variety of functional changes. One such difference which has been described is an enhanced metabolism of arachidonic acid (AA) via the 5-lipoxygenase (5-LO) pathway in alveolar macrophages (AM) as compared to PBM. In order to elucidate a possible mechanism for this difference, we compared the metabolism of endogenously released AA mobilized by agonists and of exogenously supplied fatty acid in adherent autologous PBM and AM obtained from six normal subjects. Exogenous AA was metabolized to larger amounts of both cyclooxygenase (CO) and 5-LO products by PBM as compared with AM. Although the two cell types released similar amounts of endogenous AA in response to ionophore A23187, marked differences in the pattern of its metabolism were observed. In PBM, a large proportion of released AA remained unmetabolized, and that which was metabolized was converted predominantly to CO products. In contrast, arachidonate released by AM was efficiently metabolized, predominantly via the 5-LO pathway. Similar results were obtained when cells were stimulated with the particulate zymosan, with PBM synthesizing mainly CO and AM, mainly 5-LO eicosanoids. In addition, culture of PBM for up to 5 days in an aerobic environment did not alter their response to A23187 stimulation. These results suggest that the lesser 5-LO metabolism by PBM than AM is not explained by lesser phospholipase or 5-LO activities, but rather a compartmentalization of the endogenous AA deacylated by phospholipase and the 5-LO enzyme in the PBM. The acquisition of the capacity to metabolize endogenous AA to large quantities of 5-LO products as mononuclear phagocytes differentiate in the lung may equip them with the ability to mount an inflammatory response in the alveolar space.