Further studies on generation of macrophages in in vitro cultures of mouse spleen cells and its inhibition by the capsular polysaccharide of Klebsiella pneumoniae.

Although the number of macrophages detected in cultures of mouse spleen cells at the start of the culture was very small, it markedly increased during further incubation. Macrophages were generated not only from the glass-adherent cell fraction of spleen cells, but also from the nonadherent cell fraction obtained after removal of adherent cells either by incubating in glass petri dishes or by passing through a glass bead column. The generation of macrophages from the nonadherent cell fraction occurred even when it was separated as late as 48 hr after the start of the culture. The phagocytic activity of macrophages newly generated from the nonadherent cell fraction was relatively weak, but it was activated during further incubation. Based on these results, the maturation process of macrophages can be divided into at least the following four stages; glass-nonadherent nonphagocytic precursor cells, glass-adherent nonphagocytic precursor cells, immature macrophages with low phagocytic activity, and mature macrophages with full phagocytic activity. The addition of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) to cultures of spleen cells markedly suppressed the generation of macrophages. The suppressive effect of CPS-K depended on its dosage, and the minimum concentration of CPS-K showing a definite effect was 0.05 μg/ml. CPS-K inhibited further generation of macrophages in either the nonadherent or adherent cell fraction at any time after the start of the culture. The suppressive effect of CPS-K on the generation of macrophages could not be reversed by simple washing of spleen cells which had been kept in contact with CPS-K for 3 hr. There was no evidence which showed that CPS-K exhibited direct cytotoxic effects on spleen cells in the culture.     

Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial and smooth muscle cells

Freshly isolated neonatal porcine aortic tissue (smooth muscle with or without endothelium present) produced approximately 30 ng/mg wet tissue of 6-oxo-prostaglandin F1 alpha (the stable hydrolysis product from prostacyclin) and approximately 15 ng/mg of prostaglandin E2, as measured by radioimmunoassay after 24 h incubation in culture medium. Primary cultures of porcine endothelial and smooth muscle cells (isolated by enzymic digestion of aortic tissue) exhibited the same pattern of prostaglandin production, but absolute values were greater than for fresh tissue, particularly in the case of endothelium. Subcultures of endothelium produced smaller amounts of prostaglandins, although the pattern remained similar. In contrast, subcultures of smooth muscle cells produced a greater total amount of prostaglandins than did primary cultures, and the main product was prostaglandin E2. Experiments with [14C] prostaglandin H2 or [14C]arachidonic acid confirmed that aortic tissue, cultured endothelium, and primary cultures or aortic smooth muscle cells synthesized prostacyclin, and demonstrated that subcultured smooth muscle cells enzymically isomerised prostaglandin H2 to prostaglandin E2. Kinetic studies showed that prostaglandin production by cultured vascular cells was transiently increased by subculture or changing the growth medium, and that production per cell declined with increasing cell density. The change in pattern of prostaglandin production during culture was shown to be due to a rapid decline in the rate of prostacyclin production (which apparently began immediately after tissue isolation), together with a more gradual rise in prostaglandin E2 production. These results indicate that the amounts and ratios of prostaglandins produced by vascular endothelial and smooth muscle cells are greatly affected by the conditions used to isolate and culture the cells; vascular cells in vivo may similarly alter their pattern of prostaglandin production in response to local changes in their environment.     

Mechanisms of strain-dependent development of mast cells from mouse splenocytes

Mast cell development from spleen cells was not triggered by prostaglandin E1 (PGE1) or dibutyryl cAMP (db-cAMP) during a 12 day culture when the spleen cells were obtained from C57BL/6N and DBA/1 mice, but mast cells did develop when the spleen cells were obtained from C3H/HeN, BALB/c and ICR mice. A lack of endogenous IFN-gamma in the initial 2 days of the culture period was responsible for the failure. This was confirmed by adding neutralizing anti-IFN-gamma antibody and rIFN-gamma to the cultures and by determining IFN-gamma levels in the spleen cell cultures. Th1 cells in the spleens of C57Bl and DBA/1 mice were much more sensitive to PGE1 and db-cAMP than Th1 cells from other inbred mice strains, and consequently, IFN-gamma production was inhibited in spleen cell cultures of C57BL and DBA/1 mice on addition of PGE1 or db-cAMP. Furthermore, the different sensitivities of Th1 cells to PGE and db-cAMP were dependent on the different levels of IL-12 p40 monomers or homodimers in the spleen cell cultures. As the endogenous specific inhibitors of IL-12 p70 (heterodimers of p40 and p35), large amounts of IL-12 p40 monomers or homodimers in the spleen cell cultures of C57BL and DBA/1 mice enhanced the ability of PGE1 and db-cAMP to inhibit IFN-gamma production by antagonizing the activity of IL-12 heterodimers. These results indicate that the strain-dependent development of mast cells from mouse splenocytes is related to endogenous IFN-gamma levels, which are regulated by PGE, db-cAMP, IL-12 p70 and IL-12 p40.     

Prostaglandin production by type II alveolar epithelial cells.

Prostaglandin production was studied in fetal and adult type II alveolar epithelial cells. Two culture systems were employed, fetal rat lung organotypic cultures consisting of fetal type II cells and monolayer cultures of adult lung type II cells. Dexamethasone, thyroxine, prolactin and insulin, hormones which influence lung development, each reduced the production of prostaglandin E and F alpha by the organotypic cultures. The fetal cultures produced relatively large quantities of prostaglandin E and F alpha and smaller quantities of 6-keto-prostaglandin F1 alpha and thromboxane B2. However, prostaglandin E2 production was predominant. In contrast, the adult type II cells in monolayer culture produced predominantly prostacyclin (6-keto-prostaglandin F1 alpha) along with smaller quantities of prostaglandin E2 and F2 alpha. The type II cells were relatively unresponsive to prostaglandins. Exogenously added prostaglandin E, had no effect on cell growth, and only a minimal effect on cyclic AMP levels in the monolayer cultures.     

Cell growth and differentiation in vitro in mouse macrophages transformed by a tsA mutant of simian virus 40. I. Cellular response in proliferative and phagocytic activities to the shift of temperature differs depending on the culture state in mouse bone marrow cells transformed by the tsA640 mutant of simian virus 40

It was shown previously that mouse bone marrow cells transformed by simian virus 40 (SV40) show a reversible cell density-dependent phenotypic transition between the nonmacrophage (rapidly growing) and the macrophage (stationary) states; cells in low-density cultures are in the growing phase, express SV40 T antigen strongly as revealed by immunofluorescence, and lose typical macrophage properties such as immune phagocytosis; whereas cells in high-density cultures are in the stationary (nongrowing) phase, express SV40 T antigen weakly, and recover their macrophage properties (Takayama, 1980). In the hope of clarifying the relationship between T antigen, cell growth, and macrophage-specific cellular function, we examined the behavior at 33 and 39 degrees C of mouse bone marrow cells transformed by an SV40 gene A mutant (tsA640) whose mutation renders the molecular weight of 90K (large) T antigen temperature sensitive. The results presented in this paper suggest that functional large T antigen is required for cells in the stationary phase to initiate multiplication when transferred at lower density and is not necessary for a majority of them to maintain the nongrowing state (viability) at both high and lower cell densities, whereas it is required for cells in the growing phase to keep multiplying without losing their viability. The results also suggest that the functional large T antigen does not play a direct role in maintaining the cells as either phagocytic or nonphagocytic. It is also suggested that the physiological or tsA mutation-mediated arrest of growth may or may not be accompanied by induction and/or maintenance of cellular phagocytic activity depending on the culture state.     

Defective Fc-mediated phagocytosis in gamma-irradiated mouse peritoneal macrophages

Ingestion of bovine red blood cells opsonized with IgG, by irradiated and control cultures of mouse peritoneal macrophages, was monitored at various times following exposure to 7.5-20 Gy of 60Co. Radiation produced decreases in the percentage of phagocytic cells and reduced the phagocytic index of the macrophages at 6-10 days post-irradiation. Only a small decrease in the phagocytic index of irradiated cultures was noted on day 3 post-irradiation. Cell survival as monitored by cell number and lactic dehydrogenase release as well as the levels of beta-glucuronidase and lysozyme were less sensitive to radiation exposure than was the phagocytic ability of the cultures. Addition of 8-bromo-3',5'-cyclic adenosine monophosphate and prostaglandin E2 to cultures increased the phagocytic ability of both irradiated and control cultures but did not abolish the deficit produced by radiation. The data indicate that in vitro radiation exposure produces time-dependent changes in the ability of mouse peritoneal cells to ingest IgG coated red blood cells.     

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.     

Comparative effects of cytosine arabinoside and a prostaglandin E2 antagonist, AH6809, on chondrogenesis in serum-free cultures of chick limb mesenchyme

The present study was designed to compare effects of an established inhibitor of cell proliferation and growth, cytosine arabinoside (Ara C), with that of a prostaglandin E2 (PGE2) antagonist, AH6809, on chondrogenesis in cultured mesenchyme derived from stage 25 chick limb buds. Continuous treatment of cell cultures with 10(-4) M AH6809 prevented completely the twofold increases in DNA content of control cultures which occurred between Day 1 and Day 5 of culture and also produced 90% inhibition of chondrogenesis occurring in control cultures during this same period. Treatment of cells with Ara C (0.1-0.5 microgram/ml) produced equivalent inhibition of DNA content during the same time period; however, chondrogenesis, as evaluated on Day 5 of cell culture, remained at approximately 90% of control cultures. These results indicate that the inhibitory effect of PGE2 receptor blockade on cell growth in these cultures cannot account for the potent inhibitory effects observed on differentiation of cartilage and provide further evidence in support of the notion that PGE2 plays an important initiating role in the process of chondrocyte differentiation within limb mesenchyme.     

Prostaglandin-independent effects of aspirin on cell cycle and putrescine synthesis in human colon carcinoma cells

Aspirin consumption has been reported to be able to reduce colorectal cancer risk in humans and in animal models of colon carcinogenesis. Although the mechanism involved in such an effect is not yet clear, both prostaglandin-dependent and -independent effects have been proposed. Using HT-29 Glc(-/+)cells, which originate from a human colon adenocarcinoma, we demonstrated in this study a dose-dependent effect of millimolar concentration of aspirin on cell growth that was concomitant with a rapid accumulation of the cells in the G0/G1 phase, followed by an accumulation in the G2/M phase and by a minor increase in the proportion of cells undergoing nuclear condensation. Cell membrane integrity and cell release into the culture medium were not affected by this treatment. The aspirin effects were apparently unrelated to prostaglandin biosynthesis inhibition, since although these cells were found to express high levels of cyclooxygenase 1 (COX-1) and low levels of COX-2 proteins, they did not produce any measurable net amounts of prostaglandins, based on both utilization of radiolabelled arachidonic acid and the radioimmunoassay of prostaglandins E2 and F2 alpha. In contrast, we identified polyamine biosynthesis as a cellular target of aspirin, since the treatment of HT-29 Glc(-/+) cells with aspirin reduced the flux of L-ornithine through ornithine decarboxylase, an effect that could not be explained by an acute action of the drug on the ornithine decarboxylase catalytic activity. Since polyamine biosynthesis is strictly necessary for HT-29 cell growth, our data suggest that reduced flux through ornithine decarboxylase may participate in the antiproliferative activity of aspirin towards colonic tumoral cells. It is concluded that in HT-29 Glc(-/+) cells that are not functional for prostaglandin production, aspirin can affect cell growth, cell cycle, and polyamine biosynthesis without affecting cell membrane integrity.     

Spontaneous proliferation in unfractionated spleen cell cultures: autologous mixed-lymphocyte reactions (AMLR) which can be differentially regulated by prostaglandins and lymphokines

The present studies were undertaken to define the contribution of the autologous or syngeneic mixed-leukocyte reactions (AMLR/SMLR) to the cellular proliferation observed in unfractionated spleen cell cultures. Proliferation was studied in whole, untreated 6-day murine spleen cell cultures supplemented with syngeneic serum. These cultures exhibited relatively low but significant levels of cellular proliferation as measured by uptake of radioactive thymidine ([3H]TdR). Treatment of spleen cells with monoclonal anti-Thy 1.2 antibody and complement before culture, the addition of specific anti-I-A monoclonal antibodies to the cultures or removal of Ia+ adherent cells before initiation of culture all inhibited the proliferative response significantly. Thus, the autologous proliferation of untreated and unfractionated spleen cells manifests the main characteristics of the AMLR/SMLR, namely, its dependence on T (responder) and Ia+ (stimulator) cells and specific inhibition by anti-I-A antibodies. A marked augmentation in cellular proliferation was observed in unfractionated spleen cell cultures treated for the initial 24 hr of culture with 5 X 10(-6) M indomethacin, an inhibitor of prostaglandin synthesis. Conversely, the addition of 7 X 10(-9) M prostaglandin E1 (PGE1) to these cultures depressed cellular proliferation. This suppression of autologous splenic cell proliferation induced by PGE1 could be partially reversed by the addition of concanavalin A-induced lymphokine (LK) preparations early in the culture. These findings indicate that (a) the proliferation of unfractionated spleen cell cultures occurring in the absence of exogenous stimulatory signals is due largely to an ongoing AMLR, and (b) biologically active mediators with opposing influences, namely, prostaglandins and immunostimulatory LK, participate in the regulation of the AMLR.     

Effect of phorbol ester on prostaglandin regulation of proliferation in rabbit endometrial cells

We have proposed that two of the endogenously synthesized endometrial prostaglandins, prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1), play a regulatory role in growth control of the endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used here to examine the effects of the tumor-promoting, diacylglycerol mimicking, phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the prostaglandin control of cell proliferation. TPA treatment of these cultures results in: a decrease in control levels of proliferation and complete inhibition by TPA of PGF2 alpha stimulated DNA synthesis; a reduction in [3H]PGF2 alpha binding with short term treatment but an increase to above control binding level with long term treatment; an inhibition of the normal PGF2 alpha stimulated inositol polyphosphate synthesis; and a small increase in accumulation of PGF2 alpha in the culture media. Furthermore, in this culture system, TPA does not down regulate [3H]PGE1 binding; it does not alter the normal PGE1 stimulation of cAMP synthesis; and it has no effect on the normal endogenous PGE1 synthesis by these cultures. The above results are consistent with our previous observations that PGF2 alpha works through the intracellular messengers inositol polyphosphate/diacylglycerol whereas PGE1 works through cAMP.     

Prostaglandins and cyclic AMP stimulate creatine kinase synthesis but not fusion in cultured embryonic chick muscle cells

Cyclic AMP levels have been measured in cultures derived from 12-day-old chick embryonic muscle. A rise in concentration was found after the onset of myoblast fusion. Cells cultured at a medium Ca²⁺ concentration of 0.1 μM did not fuse and exhibited only a small rise in cyclic AMP concentration during culture. Addition of 1.4 mM Ca²⁺ to these cells after 50 h in culture caused rapid, synchronous fusion with a concomitant rise in cyclic AMP levels. Indomethacin, an inhibitor of prostaglandin synthesis, did not inhibit fusion, but inhibited the rise in cyclic AMP concentration. Indomethacin-treated cultures exhibited lower creatine kinase levels, though no change in the ratio of the three isoenzymes was observed. Addition of prostaglandins E₁ and E₂ to indomethacin-treated cultures overcame this inhibition. We propose that prostaglandin synthesis is a consequence of the stimulation of myoblast fusion and that via cyclic AMP it stimulates protein synthesis.     

Prostaglandin production by phagocytic cells of the mouse thymic reticulum in culture and its modulation by indomethacin and corticosteroids

The production of prostaglandins by phagocytic cells of the thymic reticulum in culture (P-TR) was studied by using high pressure liquid chromatography and radioimmunoassay. Radioimmunologic determinations showed that thromboxane B2 (TXB2), prostaglandin E2 (PGE2), and 6-keto-prostaglandin F1 alpha (6 keto-PGF1 alpha) were the major compounds released into the culture medium, whereas prostaglandin F2 alpha (PGF2 alpha) was only a minor component. Indomethacin and dexamethasone exerted a similar pattern of differential inhibition of the secretion of prostanoids. PGE2 and 6-keto PGF1 alpha productions were markedly decreased by these anti-inflammatory drugs, whereas those of TXB2 and PGF2 alpha were not or were only slightly affected. Experiments performed with an antiglucocorticoid compound (RU 38486) showed that the steroid-induced inhibition of prostanoid secretion is a classical receptor-mediated action. These results demonstrated that phagocytic cells of the thymic reticulum, which resemble the thymic interdigitating cells, produce several types of prostaglandins. Because it has been described that P-TR regulate thymocyte proliferation in vitro via the secretion of both interleukin 1 and PGE2, these results suggest that anti-inflammatory agents may be able to modulate the thymic microenvironment and, consequently, thymocyte proliferation.     

Opposing effects of dexamethasone on the clonal growth of granulocyte and macrophage progenitor cells and on the phagocytic capability of mononuclear phagocytes at different stages of differentiation

Dexamethasone, a synthetic glucocorticosteroid, was shown to modulate the colony-stimulating factor-dependent clonal growth of myeloid progenitor cells in semisolid agar cultures, enhancing the formation of granulocyte colonies (50–100%) and suppressing the formation of macrophage colonies (75–97%). Modulation of the pattern of myeloid colony formation by dexamethasone (12–125 nM) was brought about when the steroid was administered to 6-day cultures at the time of culture initiation and up to 72 hr later. Dexamethasone inhibited myeloid cell proliferation when administered to 5-day liquid cultures at culture initiation and up to 96 hr later. Dexamethasone (12–250 nM) also enhanced the phagocytic activity of bone marrow-derived mononuclear phagocytes toward heat-killed (HK) yeast cells (up to 100%) and IgG-coated sheep red blood cells (up to 60%). Enhancement of the phagocytic capability depended critically on the stage in culture at which dexamethasone was administered. Exposure to dexamethasone for 28 hr up to 96 hr of 96-hr cultures of bone marrow cells did not lead to a modulation of phagocytic activity of the developing mononuclear phagocytes. The presence of dexamethasone during the critical period of 96 hr to 120 hr after culture initiation led to an enhanced phagocytic capability, which was statistically significant already 12 hr after the administration of the glucocorticoid. Dexamethasone induced an enhanced phagocytic activity when administered at any time after culture initiation provided that it was in culture during this critical period. When added at 120 hr of culture, dexamethasone no longer enhanced the phagocytic capability of mononuclear phagocytes and when added later than 156 hr of culture suppressed it. Dexamethasone also suppressed (up to 68%) the phagocytic capability of resident and elicited peritoneal macrophages. The results suggest that glucocorticoids shift the balance of granulocyte vs. macrophage formation at early stages of precursor cell differentiation. Reduction in mononuclear phagocyte growth and enhancement of its phagocytic capability might reflect accelerated differentiation/maturation steps. The inhibitory effect of dexamethasone on macrophage formation and on the phagocytic capability of mature mononuclear phagocytes and peritoneal macrophages might be a relevant aspect of the in vivo immune suppression encountered after glucocorticoid administration.     

Sertoli-cell prostaglandin synthesis. Effects of (follitropin) differentiation and dietary vitamin E.

The synthesis of prostanoids by the Sertoli cell was assessed as part of a study on the role of vitamin E in maintaining spermatogenesis. Analyses of eicosanoid synthesis from endogenous substrate were carried out using freshly isolated Sertoli-cell-enriched preparations from both pre-pubertal and adult rats fed purified diets with and without vitamin E, as well as cells carried in primary culture. Freshly isolated cells from both the immature and fully differentiated adult testes produced PGI2 (prostaglandin I2) and PGE2, but PGF2 alpha was produced only by cells of the adult vitamin E-deficient rat. Cells from adult controls synthesized PGF2 alpha after primary culture. In contrast with other hormone responses of this cell, which are refractory in the adult, FSH (follitropin) potentiated prostaglandin production by freshly isolated cells of both immature and adult rats. The FSH response of Sertoli cells from immature animals did not change after primary culture. Adult cells were refractory to the hormone after culture, but the total amounts of prostaglandins produced by these cells were 10-fold higher than by either freshly isolated or cells of the immature in culture. Analogues of cyclic AMP did not potentiate prostaglandin synthesis. However, mepacrine, a phospholipase inhibitor, blocked the FSH effect. The finding that Sertoli cells synthesize prostaglandins and FSH enhances prostaglandin production implicates a potential role for eicosanoids in spermatogenesis and suggests that vitamin E may affect intratesticular regulators.     

Elevated prostaglandin synthetase activity in methylcholanthrene-transformed mouse BALB/3T3

Cell lines transformed from 3T3 spontaneously, by radiation, or by treatment with chemical carcinogens, polyoma and SV40 virus produce up to 5 times more prostaglandins than their untransformed parent line. Several aspects of prostaglandin biosynthesis by MC5-5 and 3T3 were compared. When stimulated by serum, bradykinin, or thrombin, MC5-5 produced 2-to 5-fold more prostaglandins than 3T3. With the use of cells labeled with radioactive arachidonic acid in their cellular lipids, these higher levels were shown not to be due to increased availability of the prostaglandin precursor, arachidonic acid. Prostaglandin synthetase activity in microsomal fractions prepared from MC5-5 was 6 times higher than that of microsomes of untransformed cells. The increased prostaglandin levels produced by transformed cells therefore appear to be the result of elevated prostaglandin synthetase activity.     

Lipoprotein lipase in heart cell cultures is suppressed by bacterial lipopolysaccharide: an effect mediated by production of tumor necrosis factor

Exposure of rat heart cell cultures, consisting mainly of nonbeating mesenchymal cells, to 50 ng/ml of bacterial lipopolysaccharide (LPS) for 24 h resulted in a more than 80% reduction in lipoprotein lipase activity. The loss of enzymic activity was accompanied by a concomitant reduction in enzyme protein, as shown by immunoblotting. Addition of LPS to the culture medium resulted also in the production of tumor necrosis factor (TNF), and the fall in lipoprotein lipase in LPS-treated cultures could be prevented by an antibody to TNF. Addition of recombinant human TNF to the heart cell cultures also depressed lipoprotein lipase activity. LPS treatment of preadipocytes in culture resulted in a fall in lipoprotein lipase activity and TNF production. Since TNF is known as a macrophage product, the cultures were tested for phagocytic capacity, and only 0.2-1.3% of the cells were shown to engulf Staphylococcus albus. Immunofluorescent staining with monoclonal antibodies OX-1, which identify leukocyte common antigen, was negative, and only 0.1 +/- 0.07% of the cells were positive after staining with OX-42 antibody to iC3b receptor. Both antibodies stained more than 98% of rat peritoneal macrophages used as controls. Since LPS treatment of macrophages at numbers comparable to or exceeding the number of phagocytic cells present in the heart cell cultures did not induce measurable amounts of TNF, it is suggested that in the heart cell cultures, TNF may be produced by cells other than macrophages.     

Stability of DNA/anti-DNA complexes.

Human monocytes in culture release small amounts of prostaglandin E (PGE) into the medium. Addition of Fc fragments of IgG to human monocyte monolayer cultures results in a marked increase in PGE release; Fab fragments, monomeric IgG, and human serum albumin have no effect. An IgG1 myeloma has no effect on PGE levels but addition of the heat aggreagted protein results in a marked increase of PGE secretion. Exposure of the cells to Con A, which binds to a specific monocyte plasma membrane receptor, also results in a large increase in PGE release. The magnitude of the increase in PGE secretion produced by exposure of the monocytes to these ligands greatly exceeds the stimulation observed after the addition of antigen-activated mononuclear cell supernatants, zymosan, Sephadex beads, or endotoxin, to monocyte cultures. Prostaglandin E2 (PGE2) accounts for approximately 70% of the total prostaglandins released by stimulated cells. After addition of Indomethacin to monocyte cultures, the stimulatory effects of the ligands on PGE release are inhibited. Addition of Con A to monocyte cultures results in an increased incorporation of [3H]-arachidonic acid into PGE2. These results suggest that this ligand stimulates synthesis as well as release of this prostaglandin.     

Arachidonic acid metabolism by isolated epidermal basal and differentiated keratinocytes from the hairless mouse

The metabolism of arachidonic acid was studied using basal and differentiated keratinocytes as well as sebaceous cells isolated from hairless mice. These disassociated cells metabolized arachidonic acid predominantly to the prostaglandin H synthase products prostaglandins E2 and D2. 12-Hydroxyheptadecatrienoic acid (HHT), prostaglandin F2 alpha, thromboxane B2 and 6-ketoprostaglandin F1 alpha were also detected. Smaller amounts of the lipoxygenase products 5-, 12- and 15-hydroxyeicosatetraenoic acids (HETEs) were also detected. The major lipoxygenase product observed was 12-HETE. No leukotrienes or dihydroxy fatty acids were observed. The identity of the metabolites was established using several high-pressure liquid chromatography solvent systems. The biosynthesis of prostaglandins E2 and D2 was very rapid and was inhibited by the addition of indomethacin to the cells. The mixed population of keratinocytes and sebaceous cells were separated into enriched fractions by metrizamide gradients and elutriation techniques. The small, undifferentiated cells had high prostaglandin H synthase and 12-lipoxygenase activity. The basal cell-enriched fractions had the highest activity. With increasing differentiation of the cells, decreased biosynthetic activity was observed. These results indicate that undifferentiated keratinocytes, that is, the basal cells, may be an important source of prostaglandins and 12-HETE but are not a source of leukotrienes for the hairless mouse. It also suggests a role for keratinocyte-derived eicosanoids in the normal physiology of epidermal differentiation.     

Glucocorticosteroid regulation of prostaglandin biosynthesis in human myometrial smooth muscle cells in monolayer culture

In the present investigation, we evaluated the production of prostaglandins by human myometrial smooth muscle cells maintained in monolayer culture in the absence or presence of glucocorticosteroids. In the presence of cortisol (10(-7) M) or dexamethasone (10(-8) M), the rate of production of prostacyclin (PGI2) by these cells was decreased significantly. The glucocorticosteroid-mediated inhibition of prostaglandin production was attenuated when cortisol-21-mesylate (10(-6) M), a glucocorticosteroid antagonist, was present in the culture medium. The rate of conversion of radiolabeled arachidonic acid to radiolabeled prostaglandins as determined by use of sonicates of myometrial cells and optimal assay conditions, however, was not affected significantly by treatment with cortisol or dexamethasone in concentrations sufficient to inhibit prostaglandin formation by more than 80%. These findings are suggestive that glucocorticosteroids act in human myometrial smooth muscle cells in culture to inhibit prostaglandin formation by way of a receptor-mediated process that does not involve inhibition of enzyme activities that are involved in the biosynthesis of prostaglandins, i.e. the conversion of arachidonic acid to prostaglandin.