Overexpression of SOCS3 inhibits astrogliogenesis and promotes maintenance of neural stem cells.

To investigate the effects of suppressors of cytokine signaling 3 (SOCS3) on neural stem cell fate, stem cells were infected with an adenoviral vector expressing SOCS3. Three days later, western blot analysis and immunocytochemical analysis revealed that the protein level of MAP2 and the number of MAP2-positive cells were significantly increased in SOCS3-transfected cells, whereas the protein level of GFAP and the number of GFAP-positive cells were significantly decreased. Furthermore, promoter assay revealed a significant reduction in the transcriptional level of signal transducer and activator of transcription 3 (Stat3) in the transfected cells. In addition, the mRNA levels of Notch family member (notch1) and inhibitory basic helix-loop-helix (bHLH) factors (hes5 and id3) were significantly up-regulated 1 day after overexpression of SOCS3. Three days after transfection, the mRNA level of hes5 was significantly decreased, whereas that of notch1 was still up-regulated. Moreover, all of SOCS3-positive cells expressed Nestin protein but did not express MAP2 or GFAP proteins. These data indicate that overexpression of SOCS3 induced neurogenesis and inhibited astrogliogenesis in neural stem cells. Our data also show that SOCS3 promoted maintenance of neural stem cells.

Identification of macrophage migration inhibitory factor as a potent endothelial cell growth-promoting agent released by ectopic human endometrial cells.

The growth of endometrial cells in ectopic locations (endometriosis) is dependent on the establishment of an adequate blood supply. Neovascularization (angiogenesis) is therefore a vital step toward the progression of this disease. We first revealed the presence of a potent mitogenic activity for human endothelial cells in the culture medium of immortalized human endometriotic cells. The activity, measured by the level of [(3)H]-thymidine incorporation into the DNA of human coronary artery endothelial cells, was then purified by anion exchange high-performance liquid chromatography. Electrophoretic analysis of one of the bioactive fractions that markedly enhanced endothelial cell proliferation showed three distinct bands with apparent molecular masses of 15.8, 12.6, and 6.5 kDa. N-terminal microsequencing of an internal peptide from the 12. 6-kDa protein showed 100% homology with human macrophage migration inhibitory factor (MIF). The protein was positively identified as MIF by Western blot analysis using a specific anti-MIF antibody. Anti-MIF antibody inhibited the bioactivity found in the evaluated fraction and the conditioned medium of primary endometriotic cell cultures, and commercial recombinant human MIF displayed a high mitogenic activity for endothelial cells. Our findings reveal that MIF is released by endometriotic cells and acts as a potent mitogenic factor for human endothelial cells in vitro. This may have a considerable interest, in view of the crucial role of angiogenesis in ectopic endometrial cell growth and activity and in numerous tissues undergoing dynamic physiological changes, such as human endometrium.

The green tea extract epigallocatechin gallate is able to reduce neutrophil transmigration through monolayers of endothelial cells.

Green tea is widely used in Asia and has also become popular in Western countries. The influence of green tea extracts on leukocytes is not well understood. Leukocytes play a crucial role in the process of inflammation. They migrate from the intravascular space into the tissue to attack micro-organisms. The aim of the current study was to investigate the influence of epigallocatechin gallate on leukocyte transmigration through endothelial cell monolayers and thereby evaluate its potential role in the inflammatory process. Human umbilical vein endothelial cells were cultured on microporous membranes to achieve a monolayer. Freshly isolated neutrophils from healthy subjects were measured with a migration assay. The amount of untreated neutrophils migrating through untreated endothelial cell monolayers was used as control and set as 100%. Neutrophils and/or endothelial cell monolayers were pre-treated with epigallocatechin gallate using relevant, as well as higher and lower concentrations. The relevant plasma concentration of epigallocatechin gallate was able to significantly inhibit neutrophil migration through endothelial cell monolayers (69 +/- 6.4% SD; p < 0.05 compared to control), when both cell types (leukocytes and endothelial cell monolayer) were treated. This is similar to the situation after resorption in-vivo. Treating either neutrophils or endothelial cell monolayers alone led to significant reductions in migratory response (only neutrophils treated: 86 +/- 8.1% SD, p < 0.05; only endothelial cell monolayers: 77 +/- 6.1%, p < 0.05). In conclusion, epigallocatechin gallate was identified as a potent inhibitor of leukocyte migration through endothelial cell monolayers. The treatment of both cell types showed an additive effect. Endothelial cells seem to be more affected than neutrophils. Further clinical investigations are necessary to understand the potential clinical consequences.

Effects of low-density lipoproteins on polymorphonuclear leukocyte functions in vitro.

In the circulation, human polymorphonuclear leukocytes (PMN) are exposed to various factors, such as lipoproteins, which could alter their metabolic and functional characteristics. In this work, the effects of low-density lipoproteins (LDL) on PMN oxidative metabolism and migration were studied in vitro. LDL stimulated PMN superoxide generation. This effect lasted for 15-20 min and was concentration-dependent. Staurosporine, a potent inhibitor of protein kinase C, did not suppress this stimulating effect. The chemotactic response of PMN to formyl-methionyl-leucyl-phenylalanine and C5a was inhibited by LDL and this effect was conserved after trypsination of LDL. LDL from normolipidemic subjects were more potent than LDL from hypertriglyceridemic subjects for both effects. LDL had no effect on superoxide generation by opsonized zymosan-stimulated PMN. These data showed that PMN responses may be modified by environmental conditions such as the presence of lipoproteins.

Neurotrophic factor receptors and their signal transduction capabilities in rat astrocytes.

Until recently, astrocytes were not considered as sites for neurotrophic factor action. We show here that, both in vivo and in vitro, astrocytes express receptors for two separate families of neurotrophic factors. In the intact adult rat CNS, astrocytes express the extracellular domain of the neurotrophin receptor TrkB and, in a more restricted population, the low-affinity nerve growth factor receptor p75LNGFR. In the lesioned CNS, expression of the alpha component of the receptor for ciliary neurotrophic factor (CNTFR alpha) switches from a purely neuronal localization to cells in the glial scar at the edge of the wound. Using cultured hippocampal astrocytes as a model to address the functional status of these receptors, we have found only the truncated forms of TrkB and TrkC, which are incapable of signal transduction as measured by protein tyrosine phosphorylation or immediate early gene induction. In contrast, a fully functional CNTF receptor complex capable of signal transduction is present on cultured astrocytes. Thus, the neurotrophin receptors may act primarily to sequester or present the neurotrophins, whereas in the case of CNTF a functional response can be initiated within the astrocyte.

IL-10 inhibits the synthesis of migration inhibitory factor and migration inhibitory factor-mediated macrophage activation.

IL-10, in addition to being a cytokine synthesis-inhibitory factor, is a cytokine that exerts multiple effects on various cell types. Recombinant human migration inhibitory factor (MIF) inhibits the migration of human monocytes as well as that of guinea pig and murine macrophages. In addition, it has recently been shown to activate human monocyte-derived macrophages to suppress the growth of and/or kill both intracellular parasites and extracellular tumor targets in vitro and to have adjuvant activity in vivo. In this study, we examined the interactions between IL-10 and rMIF. We demonstrate that IL-10 reduces the production of MIF from T cells and abolishes rMIF-mediated migration inhibition of human monocytes. Incubation of IL-10 together with rMIF diminishes rMIF-induced intracellular killing of Leishmania donovani by human monocyte-derived macrophages and inhibits nitric oxide production and nitric oxide synthase activity by murine macrophages.

Cellular sensitivity to native type IV collagen in primary biliary cirrhosis.

We examined 60 patients with various chronic liver diseases for cellular sensitivity to native human type I and IV collagens using an in vitro leucocyte migration inhibition test. Mononuclear cells from 7 (33%) of 21 patients with chronic active hepatitis, 14 (52%) of 27 patients with liver cirrhosis and 11 (92%) of 12 patients with primary biliary cirrhosis exhibited cellular sensitivity to type IV collagen, although cells from almost all patients responded to type I collagen. None except one for type I collagen of 25 normal controls showed positive response to both collagens. In chronic active hepatitis and liver cirrhosis, cellular sensitivity to type IV collagen was significantly lower than to type I collagen (p < 0.01). Patients with primary biliary cirrhosis showed significantly higher cellular sensitivity to type IV collagen when compared to patients with other chronic liver diseases (p < 0.01). Cellular sensitivity to type IV collagen was significantly correlated with serum levels of the 7S domain of type IV collagen in all 85 subjects (r = +0.462, p < 0.001). These findings suggest that cellular sensitivity to type IV collagen as well as to type I collagen exists in chronic liver disease, especially in primary biliary cirrhosis, and may reflect the accelerated metabolism of the perisinusoidal and peribiliary basement membranes.

Interference of a neutrophil recruitment inhibitory factor upon the accumulation of inflammatory cells and airway hyperreactivity in sensitized guinea-pigs after intranasal antigen challenge.

1. A neutrophil recruitment inhibitory factor (NRIF) recovered from the crude supernatant of lipopolysaccharide (LPS)-stimulated macrophages inhibited neutrophil migration following both intratracheal and intravenous administration of LPS, but did not alter the pattern of leukopenia/leucocytosis induced by intravenous LPS. 2. The correlation between airway infiltration by inflammatory cells and hyperreactivity in lungs from actively sensitized and challenged guinea-pigs was investigated by use of NRIF. 3. Increased eosinophil counts were found in the bronchoalveolar lavage fluid from guinea-pigs sensitized with 10 micrograms ovalbumin and challenged at day 14 by the intranasal administration of the antigen. The increase was evident 5 h after challenge and persisted at 24 h. Neutrophil numbers were also increased at this time. Pretreatment with NRIF suppressed the leucocyte increase in the bronchoalveolar lavage fluid. 4. Bronchoconstriction and histamine release induced by 3 ng PAF injected into the isolated lungs were increased in challenged guinea-pigs as compared to sensitized but unchallenged controls. Pretreatment of the animals with NRIF did not interfere with this response, but significantly reduced the bronchoconstriction induced by ovalbumin injection. 5. Even though the increased number of inflammatory cells in bronchoalveolar lavage and airway hyperresponsiveness were concomitant, NRIF inhibited cellular infiltration but failed to alter airway hyperreactivity to PAF, demonstrating that these events may occur independently. Conversely, the inhibition of antigen-induced bronchoconstriction by NRIF suggests that this response is dependent upon the emigration of granulocytes.

Human recombinant migration inhibitory factor activates human macrophages to kill tumor cells.

A recombinant form of human migration inhibitory factor (rMIF) obtained from COS-1 cells transfected with MIF-specific cDNA is able to activate cultured human peripheral blood monocytes and monocyte-derived macrophages, in a dose-dependent manner to become cytotoxic for tumor cells in vitro. The cytotoxicity exhibited by macrophages treated with rMIF is > or = 30% above that of cells incubated with control supernatants or with media and peaks 72 hr after the addition of tumor targets. rMIF also induces macrophages to produce tumor necrosis factor (TNF-alpha) and interleukin-1 beta (IL-1 beta). These results demonstrate that rMIF is able to modulate macrophage functions and plays a role in cell-mediated immune response.

Role of calcium in the activation of neutrophils by leukocyte inhibitory factor (LIF).

We investigated the role of calcium in the activation of neutrophils (PMN) by the human lymphokine leukocyte inhibitory factor (LIF). Using the fluorescent probe fura 2, we demonstrated that LIF (1/2-2 units) induced a small but significant dose-dependent increase in intracellular calcium in the presence of both calcium-containing (71 nM to 158 nM) and calcium-free (53 nM to 144 nM) buffer solutions. Thus, LIF is able to release calcium from both membrane channels and from intracellular stores. However, increases in intracellular calcium were not due to a release of Ca+2 from membrane stores, as shown by the inability of LIF to diminish fluorescence of the membrane-bound calcium probe chlortetracycline. In contrast to formyl-methionyl-leucyl-phenylalanine (fMLP), maximal calcium fluxes caused by LIF were of a much lower magnitude and not observed until 10-15 min after its application. The importance of calcium to the metabolic effects of LIF was demonstrated by the abilities of the calmodulin inhibitor trifluoroperazine (51.5% inhibition) and the extracellular calcium chelator EGTA (50.5% inhibition) to suppress LIF-mediated degranulation. The intracellular calcium chelator fura 2 also significantly inhibited LIF-mediated degranulation (61.8% inhibition). However, inhibition of the release of calcium from intracellular stores by TMB-8, had no effect on LIF-mediated degranulation. These data suggest that the ability of LIF to activate PMN is dependent on the availability of intracellular calcium and that this calcium is primarily derived from the slow influx of calcium from the extracellular pool.

Lymphokine effect upon the erythrocyte antibody uptake by human monocytes.

Preincubation of human monocytes with different amounts of human lymphokine at 37 degrees C dose dependently increased the uptake of EA cells at both 37 degrees C and 4 degrees C. Phenylmethanesulphonyl fluoride (PMSF), an inhibitor of serine esterases, inhibited the process. It seemed that the serine esterase and not the gamma interferon component of the lymphokine played the main role in the phenomenon.

Inhibitory effects of shale oils (Ichthyols) on the secretion of chemotactic leukotrienes from human leukocytes and on leukocyte migration.

Ichthyols are sulfated shale oils with well-known anti-inflammatory effects in dermatologic diseases. Their possible mechanisms of action were studied by measuring chemotactic factor release from peripheral human leukocytes in vitro. Ichthyols caused no release of such factors by themselves but inhibited ionophore-induced release. After elution of the cell supernatants on reverse-phase high-pressure liquid chromatography, followed by analysis of the fractions in the chemotaxis assay and the radioimmunoassay, Ichthyols caused a reduction of lipids at marker positions for leukotriene B4 (LTB4) and 20-COOH-LTB4. The inhibition was also evident in the LTB4 radioimmunoassay, was dose- and time-dependent, and occurred in noncytotoxic concentrations of the agents. Ichthyols also inhibit the chemotactic response of neutrophils toward LTB4 and the unstimulated migration of cells. These inhibitory effects of Ichthyols on secretion of chemotactic arachidonate metabolites from leukocytes and on cell migration provide a plausible explanation for their anti-inflammatory activity.

[Leukocyte migration inhibiting factor in preparations of alpha and gamma interferons]. / Faktor, ingibiruiushchii migratsiiu leikotsitov v preparatakh al'fa- i gamma-interferona.

High titres of leukocyte migration inhibition factor (LMIF) activity were found in native and partially purified preparations of human alpha- and gamma-interferon. In the course of chemical purification of interferon up to 98% of LMIF was lost. The highest content of LMIF was in gamma-interferon preparations, while recombinant alpha 2-interferon (reaferon) showed no LMIF activity.

[Stimulation of phagocytic function by factors inhibiting leukocyte and macrophage migration in humans]. / Stimuliatsiia fagotsitarnoi funktsii faktorami, ugnetaiushchimi migratsiiu leikotsitov i makrofagov u cheloveka.

Fractions containing macrophage migration inhibition factor (MIF) and leucocyte migration inhibition factor (LIF) were obtained using Sephadex G-200 filtration from supernatant fluids of human lymphocyte cultures stimulated by PHA. The fractions were tested for the ability to affect migration and phagocytic activity of target cells. Peripheral blood leucocyte migration capacity was inhibited by the fraction with the molecular mass of 60,000-70,000 D (LIF), while migration activity of mouse peritoneal exudate cells was suppressed by the fraction with the molecular mass of 20,000-30,000 D (MIF). MIF- and LIF-containing fractions increased almost three-fold Fc-receptor-mediated phagocytic activity of neutrophils.

The effect of selenium deficiency in goats on lymphocyte production of leukocyte migration inhibitory factor.

The effect of dietary selenium on caprine leukocyte migration inhibitory factor (LMIF) production was examined in vitro using lymphocytes from goats fed a diet deficient in selenium. Selenium deficiency was determined by decreased plasma glutathione peroxidase (GSH-Px). The ability of peripheral blood lymphocytes to produce LMIF induced by concanavalin A (Con A) was significantly (P less than 0.05) inhibited when cells from selenium-deficient and selenium-adequate goats were compared. In contrast, no significant (P greater than 0.05) differences were found between lymphocytes from selenium-deficient and selenium-adequate goats for Interleukin-2 (IL-2) production and blastogenesis induced by Con A. These data suggest that selenium deficiency may selectively impair LMIF production and hence the ability of lymphocytes to modulate neutrophil migration.

Mononuclear phagocytic cells in peritoneal exudates of guinea pigs: a comparison of inducing agents.

Peritoneal macrophages are used for immunologic assessment of the lymphokine, migration inhibitory factor (MIF). For this purpose, these cells are induced intraperitoneally in animals with inflammatory agents such as mineral oil (MO) and peptone water (PW). Purpose of the present study was two-fold: To assess the changes in biologic properties of guinea pig macrophages induced peritoneally with MO or PW as compared to control cells after administration of normal saline (NS); and to examine the suitability of induced macrophages to respond to MIF in vitro. Peritoneal exudate cells were harvested from guinea pigs 7 days following intraperitoneal injection of MO, PW or NS. They were enumerated in hemocytometers, their differential counts determined by Wright's stain and morphologic characteristics assessed by scanning electron microscopy. Functional activation of peritoneal macrophages was determined by lysosomal enzyme functions, as well as by yeast phagocytosis in vitro. Random cell migration and responses to migration inhibitory factor (MIF) were determined in Mackaness chambers. Mineral oil injection resulted in significantly higher yield of peritoneal macrophages. Greater than 70% of peritoneal exudate cells were macrophages in all three groups. Spread out structures and ruffled borders were seen in electron micrographs of MO induced cells. Such structures were less evident in PW induced cells and were absent in controls. Acid phosphatase (ACP) and beta-N-acetylglucosaminidase (B-NAG) activities as well as yeast phagocytosis significantly increased in MO and PW induced cells. Random migration and responses to MIF in Mackaness chambers remained comparable in the three experimental groups.

A reappraisal of the macrophage electrophoretic mobility (MEM) test for the measurement of lymphokine activity.

Using a cell electrophoretic apparatus, which was sensitive in detecting small changes in electrophoretic mobility (EPM), the macrophage electrophoretic mobility (MEM) test was investigated as a routine method for detecting lymphokine activity. Electrophoretic analysis of guinea-pig macrophages revealed 2 main subpopulations, one with an EPM of 0.90 micron cm s-1 V-1 (fast) and the other, an EPM of 0.83 micron cm s-1 V-1 (slow). From 23 experiments the fast and slow populations were found to consist of 90% and 10% cells, respectively. When macrophages were incubated with standard guinea-pig lymphokine preparations there was a significant decrease in the fast population with a corresponding increase in the slow population. This lymphokine induced 'slowing' of the macrophages was shown to be very reproducible. Since only 50% of macrophages of high EPM were observed to respond to lymphokine activity, it is not surprising that the MEM test has failed in the past when investigators have accepted as significant a 10-15% reduction in EPM, estimated from measurements made on only 10 macrophages. Parallel bioassays indicated that there were appreciable potency differences for macrophage slowing factor (MSF) and macrophage migration inhibition factor (MIF) activities in the lymphokine preparations used which suggest that these activities may be due to different molecular entities.