Prostaglandin production by human blood monocytes and mouse peritoneal macrophages: synthesis dependent on in vitro culture conditions

The pattern of prostaglandin synthetase products from human peripheral blood monocytes was examined. Thromboxane and prostaglandin E were found to be the major products released by monocytes/macrophages on day one of culture following cell adherence. If these cells were studied 24h after cell adherence had occurred, then thromboxane synthesis was noted to be markedly reduced and PGE was the major secretory product. A day one type pattern, i.e. high thromboxane, high PGE could be elicited from day two cultured cells if cell adherence was delayed until day two of culture. Inflammatory stimuli caused a consistent rise in PGE release from day one and day two cultures, no consistent change in thromboxane was observed. It is suggested that activation of the thromboxane synthetase pathway in monocytes and macrophages is primarily a consequence of cell adherence. Prostaglandin E and prostacyclin (PGI) appear to be the major products released in response to inflammatory stimuli. These data demonstrate that the pattern and sequence of prostaglandins synthesized are in part a function of the in vitro culture conditions, time in culture and the species studied. Further, these findings offer a possible explanation to the discrepant reports in the literature.     

Adenosine deaminase activity during in vitro culture of human peripheral blood monocytes and pulmonary alveolar macrophages

Adenosine deaminase (ADA) undergoes changes in specific activity during in vitro culture of human peripheral blood monocytes and pulmonary alveolar macrophages. Monocyte adenosine deaminase activity increases during the first 3 days of culture; after 3 days the specific activity decreases below the levels observed for freshly isolated cells. In contrast, ADA activity of pulmonary alveolar macrophages increases throughout the 14-day culture period studied. Based on pH optima, starch gel electrophoresis and gel filtration column chromatography, the changes in adenosine deaminase activity in monocyte-macrophage cultures are related to changes in the molecular form of the enzyme. Freshly isolated monocytes contain mainly ES, while at day 14, starch gel and gel filtration experiments demonstrate the appearance of EI. Human pulmonary macrophages contain primarily EI or EL; following several days in culture, there is an increase in the amount of ES present.     

Prostaglandin production by human peripheral blood monocytes changes with in vitro differentiation

To investigate the influence of in vitro culture on prostaglandin (PG) production, human monocyte-enriched peripheral blood mononuclear cells were isolated and incubated on gelatin-coated plates. On days zero, five and eleven of culture, the cells were examined microscopically and the production of PGF_2α, PGE₂, PGD₂, F metabolite (PGFM) and E metabolite (PGEM) were measured by radioimmunoassay. Differences in PG output were analyzed using the Wilcoxon and Friedman tests. Freshly isolated human peripheral blood monocytes produced mainly PGE₂. In vitro, however, PGE₂ production decreased from 196 (48–288) fmol/10⁶ cells per 3h on day zero of culture to 28 (6–51) on day eleven (p=0.04); median (range), n=7. Prostaglandin D₂ and PGEM output decreased similarly, but these differences failed to reach significance. Prostaglandin F_2α and PGFM output, on the other hand, increased from 32 and 19 fmol/10⁶ cells per 3h, respectively, on day zero of culture to 127 (p<0.05) and 58 (p=0.01) on day eleven. Changes in PG output were associated with in vitro differentiation as evidenced by changes in cellular morphology. These result suggest that differentiation of human peripheral blood monocytes in vitro is accompanied by a shift in PG output from PGE₂ and PGD₂, towards PGF_2α.     

Prostaglandin production by mouse fibrosarcoma cells in culture: inhibition by indomethacin and aspirin

Five clonal strains of mouse tumor cells (HSDM₁) synthesize and secrete large quantities (0.70-2.0 μg/mg cell protein/24 hr) of prostaglandin E₂. Five lines of control cells did not synthesize significant amounts of prostaglandins. HSDM₁ cells produce prostaglandin E₂ during both the logarithmic and stationary phases of the cell growth cycle. Prostaglandin production was inhibited by aspirin-like drugs; for example, 50% inhibition was obtained with as little as 3 × 10⁻⁹ M indomethacin. We conclude that the HSDM₁ cell system will serve as a useful model system to study prostaglandin synthesis and secretion.     

Proteoglycans synthesized in vitro by nude and normal mouse peritoneal macrophages

Peritoneal macrophages from nude mice were found to be functionally similar to 'activated' macrophages from normal mice. The objective of the present study was to characterize the proteoglycans synthesized and secreted in vitro by peritoneal macrophages isolated from nude and normal Balb/c mice and to investigate the relationship between macrophage 'activation' and changes in the proteoglycan patterns. Macrophages obtained by peritoneal lavage were seeded in Petri dishes. After 2 h incubation at 37 degrees C, the adherent cells (macrophages) were exposed to [35S]sulphate for the biosynthetic labelling of proteoglycans. After incubation, the cell and medium fractions were collected and analysed for proteoglycans and glycosaminoglycans. The glycosaminoglycans were identified and characterized by a combination of agarose gel electrophoresis and enzymatic degradation with specific mucopolysaccharidases. It was shown that 3/4 of the total 35S-labelled glycosaminoglycans were in the extracellular compartment after 24-48 h. The macrophages synthesized dermatan sulphate (68%), chondroitin sulphate (7%) and heparan sulphate (25%). Both cell and medium fractions of normal and nude mouse macrophages contained glycosaminoglycans with the same ratios, although the nude mouse macrophages synthesized 2-fold less glycosaminoglycans than the normal mouse macrophages. Lower levels of 35S-proteoglycans were also obtained from in vitro 'activated' macrophages, but the ratios of dermatan sulphate:chondroitin sulphate: heparan sulphate were altered in these cells as compared to the control. Furthermore, all the 35S-macromolecules found in the extracellular compartment of nude and normal control cells were of proteoglycan nature, in contrast to the medium fractions of 'activated' macrophages, which contain both intact proteoglycans and 'free' glycosaminoglycan chains. These results indicate that, at least as regards the proteoglycans and glycosaminoglycans, the nude mouse macrophages are not identical to the 'activated' macrophages from normal mice.     

Redistribution of the Fc receptor on human blood monocytes and peritoneal macrophages induced by immunoglobulin G-sensitized erythrocytes.

The Fc receptor is a plasma membrane component exhibiting an affinity for the C gamma 3 domain of certain subclasses of immunoglobulin G. Using anti-Rho (D)-sensitized red cells (EA) in a slide rosette technique, we have demonstrated a translational mobility and polar redistribution of this receptor on the human blood monocyte and peritoneal macrophage. These cells, isolated from venous blood and malignant ascites and identified histochemically, showed a time- and temperature-dependent receptor capping defined by binding six or more EA confined to the cell half-perimeter. Caps formed in serum were mainly extreme caps in which bound EA appeared as a morula contiguous with the adherent cell. At 21 degrees C and 37 degrees C in serum 80% live rosettes formed caps; virtually none formed at 4 degrees C and about 25% were seen in PBS at 21 degrees C. Similarly, 10% extreme caps in PBS and 60 and 70% in serum were seen at room temperature and 37 degrees C, respectively, suggesting a serum factor(s) was important in promoting live rosette capping. Capping was reversibly inhibited by sodium azide although inhibition was incomplete even at 0.1 M, a concentration 10-fold higher than that giving complete inhibition of lymphocyte antigen-receptor capping. Azide also induced reversion of capped rosettes to diffuse, noncapped rosettes, and to levels comparable to those seen in inhibition studies. Re-exposure to EA after rosette capping failed to increase either the proportion of cells forming rosettes or the fullness of such rosettes indicating a critical number of receptors had been capped. Live rosettes induced a spherocytosis in bound EA irrespective of capping status; such changes appeared early in PBS where capping was minimal. Dead cells bore EA as normal biconcave discs. Some rosetting EA were ultimately hemolyzed upon prolonged incubation, and erythrophagocytosis was seen in about 15% of capped rosettes at 37 degrees C.     

Activation of mouse peritoneal macrophages in vitro and in vivo by interferon-gamma

To determine the role of IFN-gamma in the activation of resident mouse peritoneal macrophages, crude macrophage-activating lymphokines were incubated with a monoclonal anti-murine IFN-gamma antibody. This treatment abolished the capacity of mitogen-induced lymphokines to enhance either H2O2 release or activity against the intracellular protozoa Toxoplasma gondii and Leishmania donovani. All macrophage-activating factor detected by these assays was also removed by passing the lymphokines over a Sepharose column to which the monoclonal anti-IFN-gamma antibody had been coupled. Therefore, pure murine rIFN-gamma was tested both in vitro and in vivo as a single activating agent. After 48 hr of pretreatment in vitro with 0.01 to 1 antiviral U/ml, macrophage H2O2-releasing capacity was enhanced an average of 6.4-fold; half-maximal stimulation was induced by 0.03 U/ml. Resident macrophages infected with T. gondii half-maximally inhibited parasite replication after 24 hr of preincubation with 0.14 U/ml of rIFN-gamma, and near complete inhibition was achieved by pretreatment with 100 U/ml. Half-maximal leishmanicidal activity was induced by 0.08 U/ml of rIFN-gamma, and 67 to 75% of intracellular L. donovani amastigotes were killed after macrophages were preincubated with 10 to 100 U/ml. Eighteen hours after parenteral injection of rIFN-gamma, peritoneal macrophages displayed a dose-dependent enhancement of H2O2-releasing capacity and antiprotozoal activity. Half-maximal enhancement required 85 to 250 U or rIFN-gamma given i.p. Peritoneal macrophages were also activated by rIFN-gamma injected i.v. and intramuscularly. These results suggest that, in the mouse model, IFN-gamma is likely to be a primary factor within mitogen-induced lymphokines responsible for activating macrophage oxidative metabolism and antiprotozoal activity, and indicate that rIFN-gamma is a potent activator of these effector functions both in vitro and in vivo. These findings provide a rationale for evaluating rIFN-gamma in the treatment of systemic intracellular infections, and indicate that murine models are appropriate for such studies.     

Prostaglandin and thromboxane production by human and guinea-pig macrophages and leucocytes.

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection. The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemia leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE2, PGD2, PGF2 alpha, TXB2 and 6-keto-PGF1 alpha. In all types studied PGE2 and TXB2 were the major products formed. The identification of PGE2 and TXB2 was confirmed by GC/MS with multiple ion monitoring. The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.     

IL-21 dependent IgE production in human and mouse in vitro culture systems is cell density and cell division dependent and is augmented by IL-10

IL-21 is known to enhance immunoglobulin production using human in vitro models. Using either PBMC or purified tonsilar B cells both stimulated with anti-CD40, IL-4+/-IL-21, this enhancement was shown to correlate with increased cell division especially for IgE and to a lesser extent for IgM and total IgG. Cell division was monitored by CFSE staining and maximum cell division was found at low initial cell plating densities. A correlation between increased cell division and IL-10-mediated enhancement of IgE production was also seen; however, increased cell division plays a smaller role with IL-10 than IL-21. This is further emphasized in that when IL-10 and IL-21 were added together there was a further synergistic increase in IgE seen, but no accompanying further increase in cell division. The mouse system was also examined for IL-21 effects as a function of cell concentration, and as in humans, IL-21 added to murine cells increased IgE production over IL-4/CD40 stimulated cells at lower cell concentrations; however, IL-21 significantly reduced IgE at higher plated cell concentrations.     

Sodium-dependent nucleoside transport in mouse lymphocytes, human monocytes, and hamster macrophages and peritoneal exudate cells.

Mouse splenocytes and hamster peritoneal exudate cells (PEC), including macrophages, were shown to contain a predominantly Na(+)-dependent and inhibitor (6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside, NBMPR)-resistant transport system for adenosine and other nucleosides. Adenosine (1 microM) was transported about equally in mouse thymocytes and human monocytes from peripheral blood by a Na(+)-dependent system and the NBMPR-sensitive facilitated diffusion system. Hamster PEC also transported inosine, tubercidin, formycin B, uridine, and thymidine in a NBMPR-insensitive manner. With the exception of formycin B, all nucleosides were phosphorylated intracellularly to varying degree, adenosine being almost fully phosphorylated. During the time course of routine experiments (30 s) formycin B was concentrated twofold over external medium levels (1 microM) without any drop-off in the transport rate. On the basis of metabolic studies it was estimated that uridine and tubercidin were also transported against a concentration gradient. Inosine, guanosine, 2'-deoxyadenosine, tubercidin, formycin B, and the pyrimidines uridine, thymidine, and cytidine (all 100 microM) inhibited transport of adenosine and inosine about 50-100%, while 3'-deoxyinosine showed weak inhibitory action. Transport of thymidine was strongly inhibited by nucleosides except by 3'-deoxyinosine. The Na(+)-dependent, active, and concentration transport system appears to be a feature of many immune-type cells, and its presence offers particular conceptual possibilities for the therapy of infections located in these cells.     

Uptake of ionic 55Fe by mouse peritoneal macrophages in vitro.

Mouse peritoneal macrophages were maintained in vitro up to 3 days and exposed to radiolabelled ⁵⁵Fe in the form of ferrous citrate, ferrous sulfate, and ferric chloride in concentrations of 3–5 γ Fe/ml. The divalent iron compounds were taken up 10–40 times more extensively per weight of iron than the trivalent iron compounds. The net uptake of ferrous citrate was linear during the first day and thereafter increased at a slower rate. Macrophages in culture for 1 week showed one-third the average uptake of freshly cultured cells during comparable periods of exposure to ferrous citrate. The iron taken up was used in the synthesis of mouse ferritin. Uptake of ferrous citrate was influenced by serum concentration in the tissue culture medium, temperature, pinocytosis and phagocytosis of both latex particles and heated rat erythrocytes. Uptake of ferrous citrate was enhanced by exposure to either sodium fluoride (5×10^?3 M), or 2,4-dinitrophenol (1×10^?5 M), but was not affected by cyanide, azide, or cycloheximide. The effect of sodium fluoride was not demonstrated when ferrous sulfate was substituted for ferrous citrate. The results reported here suggest that the ability of macrophages to take up ferrous citrate is good in freshly explanted cultures, is a temperature-dependent process, is suppressed by pinocytosis and phagocytosis, and paradoxically enhanced by certain metabolic inhibitors.     

HLA-DR expression on human peritoneal macrophages in vivo and in vitro

Qualitative, semi-quantitative (immuno-electronmicroscopy), and quantitative (radioimmunoassay) measurements were made of the in vivo and in vitro expression of HLA-DR on continuous ambulatory peritoneal dialysis (CAPD) patients' peritoneal macrophages (M phi) and on healthy persons' blood monocytes (MO). In vivo, great variation is seen in both the qualitative and (semi-) quantitative expression of HLA-DR in peritoneal M phi. After culturing for 5 to 20 h, CAPD patients' M phi with low to intermediate numbers of HLA-DR molecules per cell (25-80 x 10(3] showed a two- to threefold enhancement of HLA-DR expression. This enhancement was determined for the total peritoneal cell (PC) population and for the adherent subpopulation of peritoneal M phi and blood MO. CAPD patients whose cells initially had high numbers of HLA-DR molecules (80-110 x 10(3] showed no or only slight enhancement of HLA-DR expression when cultured.     

Prostaglandin E2 production by rat peritoneal macrophages: role of cellular and humoral factors in vivo in transfusion-associated immunosuppression

The transfusion of blood is associated with long-term immunosuppression, which has been postulated to influence immunosurveillance and cancer cell killing. The mononuclear phagocyte synthesises large quantities of PGE2, and PGE2 has been shown to inhibit the activity of a range of immunocompetent cell types. The role of mononuclear phagocyte PGE2 synthesis in transfusion-associated immunosuppression, and the elements of transfused blood which control this immunosuppression, were investigated using a transfused rat model. A significant increase in macrophage PGE2 synthesis was detected 7 days after transfusion with blood and serum. The storage of blood for 24 h increased the stimulatory activity of transfused blood. The effects of storage and serum on macrophage PGE2 synthesis were greater than effects due to genetic differences between blood donor and recipient, and the serum effects indicated that a major factor activating PGE2-mediated immunosuppression in transfused subjects may be humoral in nature.     

Effect of endotoxin on the production of colony-stimulating factor by human monocytes and macrophages

Colony-stimulating factor (CSF) is necessary for the clonal growth of human bone marrow in vitro. Human blood monocytes and macrophages produce CSF. Endotoxin was found to increase the level of CSF generated by macrophages, but had no stimulatory effect on monocytes. Several other substances known to influence the pinocytic or phagocytic activity of mononuclear phagocytes failed to enhance cellular CSF generation.     

Phagocytosis in vitro of polyethylene glycol-modified liposome-encapsulated hemoglobin by human peripheral blood monocytes plus macrophages through scavenger receptors

Liposome-encapsulated hemoglobin (LEH), a candidate for red blood cell substitute, is cleared from circulation primarily by the phagocytic system, most likely after opsonization of the vesicles by immunoproteins, particularly complement components. Although modification of LEH by polyethylene glycol (PEG) derivatives prolongs its half-life by blocking the opsonization, the half-life is still short as compared with that of red blood cell components. Therefore, this study was performed to elucidate the opsonin-independent mechanisms that regulate phagocytosis of Neo Red Cell (NRC), a PEG-modified LEH, in culture. PKH67 was used as a fluorescence marker, allowing the quantitation of the phagocytosis of NRC by peripheral blood monocytes plus macrophages. The phagocytosis of PKH67-labeled NRC was inhibited by the addition of an excess of unlabeled NRC, indicating that the phagocytosis of PKH67-labeled NRC is specific to NRC, but not to PKH67. The phagocytosis of NRC was blocked about 70% by anti-CD14, 60% by anti-CD36 and 30% by anti-CD51/61 (vitronectin receptor, alpha(v)beta3). These results provided evidence of an opsonin-independent pathway for the phagocytosis of PEG-modified LEH.     

Effects of human defensin HNP-1 on the production of tumor necrosis factor-alpha by human blood monocytes in vitro]

The influence of human defensin HNP-1 on the synthesis of tumor necrosis factor-alpha (TNF-alpha) by human blood monocytes was studied. It was shown that TNF-alpha production by human monocytes activated by SAC or PMA was augmented in the presence of HNP-1 concentrations of 10(-8)-10(-9) M. HNP-1 alone induced no synthesis of TNF-alpha. High concentration of HNP-1 (10(-4) M) was cytotoxic for human monocytes.     

Possible involvement of protein kinase C in interleukin-1 production by mouse peritoneal macrophages

Both lipopolysaccharide (LPS) and phorbol-12,13-dibutyrate (PDBu), a protein kinase C-activating phorbol ester, induced interleukin-1 (IL-1) production in mouse peritoneal macrophages. Prolonged treatment of the cells with PDBu led to the down-regulation and complete disappearance of protein kinase C. In these cells, PDBu did not increase IL-1 production, but LPS still stimulated IL-1 production although the maximum level was slightly reduced. These results suggest that protein kinase C and another unknown signal pathway are involved in LPS-induced IL-1 production.     

Uptake and utilization of human polymorphonuclear leukocyte granule myeloperoxidase by mouse peritoneal macrophages

Summary Functional myeloperoxidase contained in granules of polymorphonuclear neutrophil leukocytes or in fixed whole cells can be endocytosed by mouse peritoneal macrophages. Acquired myeloperoxidase was distributed in what we considered to be the secondary lysosomal system and, following a phagocytic stimulation, was delivered to newly formed phagosomes containing the targets.