Inhibition by glucocorticoids and choleragen of the conditional growth of poorly adherent mononuclear phagocytes of newborn hamster liver and lung (hormonal control of macrophage growth)

Conditions for in vitro growth of mononuclear phagocytes from newborn hamster liver and lung were studied. In the primary cultures of liver and lung, round cells outgrew and frequently floated off into the culture medium. They were separated from fibroblast-like cells adherent to plastic by collecting the medium. The round cells were identified as mononuclear phagocytes on the criteria of phagocytic capacity of heat-killed bacteria and IgG-coated erythrocytes, fine cell structure and cytochemistry. The phagocytes that had not been activated previously proliferated for about ten generations in F12 medium supplemented with 10% fetal calf serum depending on a growth factor produced by hamster brain, liver or lung cells. Without the factor, the cells quickly cytolysed. Mononuclear phagocytes from blood had the same characteristics of growth and cytochemistry, but had fewer IgG receptors at the cell surface than similar cells from the liver and lung. The effects of a variety of chemical compounds on the growth of the liver and lung cells were studied. Insulin stimulated their growth by 20-30%, but was not replaceable for the growth factor. Glucocorticoids, dexamethasone and hydrocortisone, inhibited the growth of the phagocytes at the physiological concentrations: 3 x 10(-9) M and 2 x 10(-8) M for 50% inhibition, respectively. Indomethacin, non-steroid anti-inflammatory reagent, at 10(-8) M to 10(-6) M gave no effect. Choleragen that increases the intracellular cyclic AMP level, inhibited the growth at a concentration as low as 5 pg/ml. These data suggest that the growth of mononuclear phagocytes is controlled not only by a growth factor produced by other cells but also by glucocorticoids.     

Contribution of different organs to increased glucose consumption after endotoxin administration

Glucose utilization of different organs (spleen, liver, ileum, kidney, skin, lung, and testis) was investigated in vivo in conscious rats 3, 24, or 48 h after treatment with 100 micrograms of endotoxin/100 g of body weight. Glucose uptake was determined by the 2-deoxyglucose technique, which was validated by demonstrating that endotoxin treatment did not alter either the intracellular retention of the phosphorylated metabolites (P-2-dGlc) of the tracer or the discrimination against 2-deoxyglucose in pathways of glucose metabolism. At 3 h after endotoxin the accumulation of P-2-dGlc was markedly increased in the liver (4.8-fold), spleen and skin (2.9-fold), lung (2.4-fold), and ileum and kidney (2.1-fold), as compared to time-matched controls. This effect was sustained in the liver at 24 and 48 h, was diminishing but still significant in spleen, ileum, and kidney, and absent in skin and lung. Accumulation of P-2-dGlc in the testis remained unchanged after endotoxin. Glucose uptake by individual organs and their contribution to whole body glucose utilization in control and endotoxin-treated rats were compared based on P-2-dGlc accumulation data. Organs rich in mononuclear phagocytes (liver and spleen) exhibited a marked and prolonged increase in glucose uptake after endotoxin. Yet the bulk of the increment in the whole body glucose disappearance rate (Rd) was due to three large tissues (skin, intestine, and muscle, accounting for more than 80% of the total P-2-dGlc accumulation in soft tissues), which showed a more moderate and transient increase in glucose utilization.     

Cytochemical localization of glucose-6-phosphatase in rabbit mononuclear phagocytes during differentiation

Cytochemical and biochemical investigations have revealed glucose-6-phosphatase (G-6-Pase) activity in Kupffer cells of the liver. To determine whether other mononuclear phagocytes are also reactive for G-6-Pase, rabbit bone marrow, blood, and alveolar macrophages were tested for G-6-Pase by a modified Wachstein-Meisel method and prepared for electron microscopy. Some mononuclear phagocytes from all three tissues were intensely reactive; others were unreactive. In promonocytes, monocytes, and alveolar macrophages, reaction product for the enzyme was localized throughout all cisternae of the endoplasmic reticulum (ER) and the perinuclear cisternae, but it was absent from the Golgi complex, lysosomes, and occasional smooth tubular channels. These results indicate that mononuclear phagocytes at all stages of development contain cytochemically demonstrable G-6-Pase and that the distribution of the enzyme is not altered during their differentiation from immature cells in the bone marrow to mature macrophages in the lung.     

Mononuclear phagocytes of rat liver and lung after intravenous introduction of suspension of magnetite nanoparticles

Changes in morphology and intraorgan localization of cells of the mononuclear phagocyte system in rat liver and lung are described at various intervals after single and multiple intravenous injection of suspension of unmodified nanoparticles of magnetite (NPM). A possible mechanism is illustrated of elimination of NPM from the rat organism with the participation of mononuclear phagocytes of liver (removal of stellar macrophages into the gastrointestinal tract through bile ducts) and lung (migration of macrophages into the bronchial tree lumen). A possibility is discussed of NPM elimination by different mechanisms, for instance, by metabolism in lysosomes by analogy with transformations of nanoparticles of different qualitative compositions described in the literature. Intravenous administration of NPM has been shown to cause changes in the morphology of rat visceral organs, the expression of these changes being amplified with a rise in the total dose of the introduced magnetite. Nanomagnetite is revealed in rat liver and lung 40 days after single intravenous introduction.     

Surfactant protein A suppresses lipopolysaccharide-induced IL-10 production by murine macrophages

Upon LPS exposure, mononuclear phagocytes produce TNF-alpha and IL-10, two cytokines with pro- and anti-inflammatory activities, respectively. We previously described that murine resident alveolar macrophages, which play a central role in the immunosurveillance of the lung alveoli, do not synthesize IL-10 in vivo or in vitro when exposed to LPS. In the present report we demonstrate that during lung inflammation induced by the intranasal administration of LPS, bronchoalveolar cells collected between days 3 and 5 are able to synthesize IL-10 when exposed to LPS. We also show that depletion of resident alveolar macrophages by an intratracheal instillation of liposome-encapsulated clodronate is followed by subsequent replenishment of the airspaces by mononuclear phagocytes. This is accompanied by the transient competence of cells for IL-10 production. The cell capacity to produce IL-10 is evident up to 3 days and then decreases. This led us to hypothesize that the alveolar environment contains a down-regulator of LPS-induced IL-10 synthesis by recently emigrating mononuclear phagocytes. We show that the surfactant protein A, an airspace protein that has known immunomodulatory activities, dramatically inhibits LPS-induced IL-10 formation by bone marrow-derived macrophages. These data show a difference between resident and inflammatory macrophages with respect to IL-10 synthesis. Moreover, this study highlights for the first time the inhibitory role of surfactant protein A in the anti-inflammatory activity of macrophages through inhibition of IL-10 production.     

CX3CR1+ lung mononuclear phagocytes spatially confined to the interstitium produce TNF-α and IL-6 and promote cigarette smoke-induced emphysema

Increased numbers of macrophages are found in the lungs of smokers and those with chronic obstructive pulmonary disease. Experimental evidence shows the central role of macrophages in elaboration of inflammatory mediators such as TNF-α and the progression toward cigarette smoke-induced emphysema. We investigated the role of CX3CR1 in recruitment of mononuclear phagocytes, inflammatory cytokine responses, and tissue destruction in the lungs after cigarette smoke exposure. Using mice in which egfp is expressed at the locus of the cx3cr1 gene, we show that alveolar macrophages increased transmembrane ligand CX3CL1 expression and soluble CX3CL1 was detectable in the airspaces, but cx3cr1(GFP/GFP) and cx3cr1(GFP/+) mice failed to show recruitment of CX3CR1(+) cells into the airspaces with cigarette smoke. In contrast, cigarette smoke increased the accumulation of CX3CR1(+)CD11b(+) mononuclear phagocytes that were spatially confined to the lung interstitium and heterogenous in their expression of CD11c, MHC class II, and autofluorescent property. Although an intact CX3CL1-CX3CR1 pathway amplified the percentage of CX3CR1(+)CD11b(+) mononuclear phagocytes in the lungs, it was not essential for recruitment. Rather, functional CX3CR1 was required for a subset of tissue-bound mononuclear phagocytes to produce TNF-α and IL-6 in response to cigarette smoke, and the absence of functional CX3CR1 protected mice from developing tissue-destructive emphysema. Thus, CX3CR1(+) "tissue resident" mononuclear phagocytes initiate an innate immune response to cigarette smoke by producing TNF-α and IL-6 and are capable of promoting emphysema.     

Effects of parathyroid hormone and calcitonin on adenylate cyclase in murine mononuclear phagocytes

Peritoneal mononuclear phagocytes elicited by thioglycollate demonstrate responsiveness to parathyroid hormone (PTH) and calcitonin (CT) which differs from that seen in the normal resident population. PTH causes a twofold stimulation of adenylate cyclase activity in elicited cells but inhibits this activity in resident cells. CT causes a greater stimulation of adenylate cyclase in elicited than in resident cells. Both CT and PTH cause an increase in cyclic AMP accumulation in cultures of elicited mononuclear phagocytes. These results indicate that cells of the mononuclear phagocyte lineage have functional receptors for both PTH and CT. This is the first biochemical evidence to support the hypothesis that mononuclear phagocytes are precursors of the bone resorbing osteoclast.     

Bacillary replication and macrophage necrosis are determinants of neutrophil recruitment in tuberculosis

We previously determined that burst size necrosis is the chief mode of mononuclear cell death in the lungs of mice with tuberculosis. The present study explored the link between infection-induced necrosis of mononuclear phagocytes and neutrophil accumulation in the lungs of mice challenged with one of four Mycobacterium tuberculosis strains of increasing virulence (RvΔphoPR mutant, H37Ra, H37Rv and Erdman). At all time points studied, Erdman produced the highest bacterial load and the highest proportion and number of M. tuberculosis-infected neutrophils. These parameters, and the proportion of TUNEL-positive cells, tracked with virulence across all strains tested. Differences in neutrophil infection were not reflected by levels of chemoattractant cytokines in bronchoalveolar lavage fluid, while interferon-γ (reported to suppress neutrophil trafficking to the lung in tuberculosis) was highest in Erdman-infected mice. Treating Erdman-infected mice with ethambutol decreased the proportion of mononuclear phagocytes with high bacterial burden and the ratio of infected neutrophils to infected mononuclear cells in a dose-dependent manner. We propose that faster replicating M. tuberculosis strains cause more necrosis which in turn promotes neutrophil recruitment. Neutrophils infected with M. tuberculosis constitute a biomarker for poorly controlled bacterial replication, infection-induced mononuclear cell death, and increased severity of immune pathology in tuberculosis.     

Human mononuclear phagocyte activation antigens

Activation of mononuclear phagocytes causes changes in plasma membrane composition that include the expression of surface antigens and receptors. Monoclonal antibody technology has made it possible to identify and characterize newly expressed surface antigens. Among these "activation antigens" is a glycoprotein, Mo3, which (among hematopoietic cells) is selectively expressed by human mononuclear phagocytes that have been exposed to inflammatory factors in vitro and in vivo. Progress toward a functional and structural analysis of Mo3 is described.     

Functional Characteristics of Enhanced Fc Receptor Expression of β2 Integrin-Deficient Bovine Mononuclear Phagocytes

Fc receptor expression, cytoplasmic Ca²⁺ signaling, chemiluminescent (CL) response, and electron spin resonance (ESR) combined with spin trapping of blood mononuclear phagocytes from control heifers and a heifer with leukocyte adhesion deficiency (LAD) were evaluated to elucidate the relationships between complement receptor type 3 (CR3) and Fc receptor expression and their functional responses. The mean fluorescence intensity of fluorescein isothiocyanate (FITC)-conjugated anti-bovine IgG bound to mononuclear phagocytes from the heifer with LAD was 1.8-fold higher than that of control heifers. The mean increments of cytoplasmic Ca²⁺ concentrations of mononuclear phagocytes from the heifer with LAD stimulated with OPZ, Agg-IgG, and PMA were 39.4 (P<0.05), 118, and 71.6% compared with those of control heifers. A 1.27-fold increase in the CL response relative to control heifers was detected when mononuclear phagocytes from the heifer with LAD were stimulated with Agg-IgG. The OPZ-induced CL response of mononuclear phagocytes from the heifer with LAD was significantly (P<0.05) decreased, whereas the PMA-induced CL response was similar to that of control heifers. The ESR spectrum of mononuclear phagocytes from the heifer with LAD was increased when stimulated with Agg-IgG, and was impaired when stimulated by OPZ compared with that of control heifers. The ESR spectrum of mononuclear phagocytes stimulated with PMA was similar in control heifers and the heifer with LAD. Fc receptors on mononuclear phagocytes from the heifer with LAD were enhanced, and their cytoplasmic Ca²⁺ signaling, CL response, and ESR-spin trapping when stimulated with Agg-IgG and OPZ appeared to be associated with enhanced Fc receptors.     

Macrophage maturation: differences in complement secretion by marrow, monocyte, and tissue macrophages detected with an improved hemolytic plaque assay

In order to examine one function of mononuclear phagocytes during maturation from bone marrow precursors to tissue macrophages, an improved hemolytic plaque assay for the detection of synthesis of the second (C2) and fourth (C4) components of C by single cells was developed. With this method, production of C2 and C4 was assessed in cell populations derived from bone marrow, blood, lung, peritoneum, and spleen. The proportion of cells producing C2 and C4 in each population varied. Approximately 10% of bone marrow cells produced C4, but not detectable C2 plaque-forming cells (PFC) were detected. Circulating monocytes yielded about 10% PFC each for C2 and C4. The proportion of C2-producing cells in tissue macrophages varied from approximately 2% in bronchoalveolar macrophages to about 45% in peritoneal and splenic macrophage populations, whereas C4 production by macrophages from lung, peritoneum, and spleen were all approximately 45%. These data suggest that differences in C biosynthesis characterize mononuclear phagocytes at different stages of maturation.     

Early structural changes in sheep lung following thoracic irradiation

Using a large animal model of radiation lung injury--the sheep exposed to bilateral thoracic irradiation--we have recently shown the development of sustained pulmonary hypertension during the first 4 weeks following radiation. This is the period prior to the onset of pneumonitis and pulmonary fibrosis. In the present study, we have examined biopsy and autopsy lung tissue from these same sheep and assessed the sequential changes in lung morphology. Six unanesthetized sheep received bilateral thoracic irradiation (a total of 15 Gy); control sheep were sham irradiated. Lung biopsy tissue was taken prior to and at weekly or biweekly intervals during the 4 weeks immediately following radiation. The lungs were also removed at autopsy for light and electron microscopic examination. Our results show early (Week 1) interstitial and progressive intraalveolar edema accompanied by endothelial and epithelial injury. A gradual increase in number of interstitial mononuclear cells was evident from Week 1, both in the lung tissue and in perivascular cuffs. The number of peripheral lung interstitial mononuclear cells was twice baseline from Week 3 and included accumulation of lymphocytes, fibroblasts, and intravascular macrophages. The increased numbers of mononuclear cells paralleled the development of chronic pulmonary hypertension, perhaps suggesting their involvement in the pathogenesis of this disease. Alternatively, it may be that increased mononuclear cell number represents a stage of lung repair.     

Antioxidative and metabolic responses to extended cold exposure in rats

In this work, we investigated whether extended cold exposure increases oxidative damage and susceptibility to oxidants of rat liver, heart, kidney and lung which are metabolically active tissues. Moreover in this study the effect of cold stress on some of the lipid metabolic mediators were studied in rat experimental model. Male albino Sprague-Dawley rats were randomly divided into two groups: The control group (n=12) and the cold-stress group (n=12). Tissue superoxide dismutase (SOD), catalase (CAT), glutathion S-transferase (GST) and glutathion reductase (GR) activities and glutathion (GSH) were measured using standard protocols. The biochemical analyses for total lipid, cholesterol, trigliceride, HDL, VLDL and LDL were done on autoanalyzer. In cold-stress groups SOD activity was decreased in the lung whereas it increased in the heart and kidney. CAT activity was significantly decreased (except liver) in all the tissues in treated rats. GST activity of cold-induced rats increased in liver and heart while decreased in the lung. GR activity was significantly decreased (except in liver) in all the tissues in cold-stressed rats. GSH level was significantly increased in the heart but decreased in the lung of animals exposed to cold when compared to controls. It was found that among the groups trigliceride, total lipid, HDL and VLDL parameters varied significantly but cholesterol and LDL had no significant variance. In this study, we found that exposure of extended (48 h) cold (8 degrees C) caused changes both in the antioxidant defense system (as tissue and enzyme specific) and serum lipoprotein profiles in rats.     

The parasitism characteristics of the causative agent of melioidosis in the host cells]

The electron microscopic study of the damaged areas of the lungs, liver, spleen and lymph nodes of guinea pigs infected with two P. pseudomallei virulent strains C-141 and 100 has revealed that this organism is a facultative intracellular parasite and exhibits tropism to reticuloendothelial cells of the body, parasitizing both in "professional" phagocytes (mononuclear, phagocytes, polymorphonuclear leukocytes) and "nonprofessional" phagocytes (capillary endotheliocytes, splenic and lymph-node reticulocytes). In the course of their intracellular development P. pseudomallei have been shown to form capsules protecting the pathogens from the unfavorable action of the protective mechanisms of the host cells.     

The effect of ceruloplasmin on hepatocyte proliferation in the regenerating rat liver

The influence of ceruloplasmin on cell proliferation in regeneration liver of the rat has been studied. Ceruloplasmin stimulates cell proliferation in regeneration liver increasing functional activity of the mononuclear phagocytes.     

Crystalline lens induction of lipid peroxidation

The level of lipid peroxidation products (LPP) was determined in the aqueous humor from the anterior chamber of patients with cataract and donor eyes. The content of LPP in senile cataract aqueous humor was shown to be significantly increased. To determine the possible mechanism of LPP increase in aqueous humor, human lenses at different stages of cataract as well as transparent human and rabbit lenses were incubated for 3 hours in 3.0 ml medium containing liposomes (0.5 mg/ml) prepared from phospholipids from the egg yolk and 0.14 M NaCl + 0.01 M TRIS-HCl buffer, pH 7.4). Corrections were made for phospholipid autooxidation. The level of LPP accumulation in the medium was determined by MDA assay. The rate of LPP production increased significantly in transparent lenses and in early senile cataract, as compared to controls and advanced (mature) cataracts. EDTA (1 mM), superoxide dismutase (114 u/sample), catalase (900 u/sample), chelated iron (III): Fe3+-ADP addition to the incubation medium depressed the level of LPP accumulation. This suggests the participation of Fe2+, O2-., H2O2 in the mechanism of LPP production in the lens. The induction of lipid peroxidation in the lens can be significant for leukotriene and prostaglandin synthesis in the eye.     

Immunodetection of cyclooxygenase-2 (COX-2) is restricted to tissue macrophages in normal rat liver and to recruited mononuclear phagocytes in liver injury and cholangiocarcinoma

It has been suggested that cyclooxygenase-2 (COX-2)-mediated prostaglandin synthesis is associated with liver inflammation and carcinogenesis. The aim of this study is to identify the cellular source of COX-2 expression in different stages, from acute liver injury through liver fibrosis to cholangiocarcinoma (CC). We induced in rats acute and “chronic” liver injury (thioacetamide (TAA) or carbon tetrachloride (CCl₄)) and CC development (TAA) and assessed COX-2 gene expression in normal and damaged liver tissue by RT-PCR of total RNA. The cellular localization of COX-2 protein in liver tissue was analyzed by immunohistochemistry as well as in isolated rat liver cells by Western blotting. The findings were compared with those obtained in human cirrhotic liver tissue. The specificity of the antibodies was tested by 2-DE Western blot and mass spectrometric identification of the positive protein spots. RT-PCR analysis of total RNA revealed an increase of hepatic COX-2 gene expression in acutely as well as “chronically” damaged liver. COX-2-protein was detected in those ED1⁺/ED2⁺ cells located in the non-damaged tissue (resident tissue macrophages). In addition COX-2 positivity in inflammatory mononuclear phagocytes (ED1⁺/ED2⁻), which were also present within the tumoral tissue was detected. COX-2 protein was clearly detectable in isolated Kupffer cells as well as (at lower level) in isolated “inflammatory” macrophages. Similar results were obtained in human cirrhotic liver. COX-2 protein is constitutively detectable in liver tissue macrophages. Inflammatory mononuclear phagocytes contribute to the increase of COX-2 gene expression in acute and chronic liver damage induced by different toxins and in the CC microenvironment.