Inhibition by cyclo-oxygenase inhibitors of interleukin-6 production by human peripheral blood mononuclear cells.

The effects of cyclo-oxygenase inhibitors on interleukin-6 (IL-6) production by human peripheral blood mononuclear cells were examined. Indomethacin and Y-9223, a novel cyclo-oxygenase inhibitor, inhibited the increases in the IL-6 level in the culture medium of both mitogen-stimulated adherent cells and non-adherent cells fractionated from mononuclear cells. Northern blotting showed that the mitogen-induced increase in the expression of IL-6 mRNA was inhibited by indomethacin and Y-9223, indicating that these agents inhibit IL-6 biosynthesis. Aspirin, ibuprofen, and phenylbutazone also inhibited IL-6 production by adherent cells stimulated with lipopolysaccharide (LPS). There was, however, no direct relationship between inhibition of IL-6 and prostaglandin E2 (PGE2) production by these agents. The addition of PGE2 corresponding to the amount produced by adherent cells stimulated with LPS slightly increased IL-6 production by unstimulated adherent cells, but to a lower level than that reached with LPS. An anti-PGE2 antibody partially blocked IL-6 production by adherent cells stimulated with LPS. These results suggest that, in addition to the inhibition of PGE2 production, other mediators including cyclooxygenase products or other action mechanisms are involved in the inhibition of IL-6 production by these drugs.     

Inhibition of hepatitis C virus adsorption to peripheral blood mononuclear cells by dextran sulfate

Summary.  Human peripheral blood mononuclear cells (PBMC) can be infected in vitro with sera of hepatitis C virus (HCV)-infected patients. We have treated pools of PBMC with dextran sulfate MW 8 000, before infection with various HCV(+) sera. When the cells were treated with dextran sulfate 10⁻³M, HCV RNA was no longer detectable after infection with all HCV genotypes tested. When the cells incubated with dextran sulfate 10⁻³M and 10⁻⁴M were maintained in culture for 4 weeks, no significant HCV replication could be observed. Our data suggest that dextran sulfate inhibits the attachment of HCV to the cell surface, since the HCV RNA detection was similar to control cells when dextran sulfate was added after infection. Accepted October 15, 1997 Received August 25, 1997     

Seasonal variation of IgE synthesis in vitro by human peripheral blood mononuclear cells

Seasonal variations in IgE antibody synthesis in vitro were studied in cultures of blood mononuclear cells (MNC) from 11 pollen allergic individuals. The IgE levels were significantly higher in two summer seasons than in the winter and spring between them. Net synthesis was confined to the summer in all but one of the patients. All the IgE in the cultures outside the pollen season represented preformed IgE which was present mainly (59%) in the monocyte fraction. Thus, preformed IgE seems to persist in monocytes at times when there is little de novo synthesis of IgE.     

Killing of Coccidioides immitis by human peripheral blood mononuclear cells.

The ability of human peripheral blood mononuclear cells (MNL) obtained from healthy donors to kill the fungus Coccidioides immitis was examined in vitro with an assay that uses a single fungal particle per well. MNL killed 25.0% +/- 3.5% of a coccidioidal arthroconidial target, compared with the 4.7% +/- 2.9% killed by polymorphonuclear leukocytes obtained from the same donors (P = 0.012). Arthroconidial killing by MNL was not dependent on donor delayed dermal hypersensitivity to spherulin. Killing of another fungal target, Candida glabrata, was not significantly different between MNL and polymorphonuclear leukocytes (P = 0.783). Depletion of monocytes from MNL with Sephadex G-10 resulted in a significant reduction in arthroconidial killing (21.4% +/- 13.6% versus 2.4% +/- 3.4%; P = 0.025), while enrichment of monocytes by Percoll density gradient centrifugation or plastic adherence resulted in significantly increased arthroconidial killing compared with that by MNL (P = 0.005 and 0.001, respectively). Killing of 96-h spherules by MNL was 7.3% +/- 3.1%, significantly less than the 21.4% +/- 2.8% killing of arthroconidia in the same experiments (P = 0.016). Incubation of MNL with human recombinant gamma interferon or tumor necrosis factor alpha did not result in increased MNL killing of coccidioidal arthroconidia under various conditions. These results suggest that MNL have an inherent ability to kill coccidioidal arthroconidia in vitro which is not dependent on prior host exposure to C. immitis. This activity appears to reside in peripheral blood monocytes.     

In vitro studies of suppressor cell function in human peripheral blood mononuclear cells.

Peripheral blood mononuclear cells (PBMC) from normal donors, pre-cultured at 37 degrees C for 24 hr before the addition of mitogen, demonstrated an enhanced proliferative response. This may be due to the loss of a subpopulation of suppressor cells during the incubation period. Still further enhancement was observed when pre-culturing was prolonged for 48 hr, while cells pre-incubated at 4 degrees C showed no increased responsiveness. Concanavalin A (Con A) pre-activated PBMC supressed the mitogen response of responder cells. More marked suppression was observed when the concentration of Con A used to induce the suppressor cells was increased. It was not possible to activate suppressor function in cells which had been kept in vitro for longer than 48 hr. These findings support the concept of the existence and function of suppressor cells, and that the suppressive influence is short-lived in vitro culture.     

Microplate hybridization for Borna disease virus RNA in human peripheral blood mononuclear cells.

We developed a simple and sensitive microplate hybridization procedure with which to identify Borna disease virus cDNA in amplified products from human peripheral blood mononuclear cells. The mean values for the positive PCR products were significant compared with those for any of the negative products, indicating that this method can be applied to rapidly diagnose a large number of clinical specimens.     

In vitro propagation of chicken anemia virus in chicken peripheral blood mononuclear cells

Keywords: chicken anemia virus; propagation; mononuclear cells; PCR.     

Esterase staining of human peripheral blood mononuclear cells.

Recently proposed esterase staining methods for the cytochemical identification of human peripheral blood monocytes and lymphocytes in our hands gave suboptimal results. Cellular purification and recommended fixation procedures appeared to decrease the esterase reactivity of leucocyte preparations. Drying for 12-18 h without further fixation of cytocentrifuge smears was found to produce minimal loss of the staining capacity for 1-naphthyl butyrate esterase in mononuclear cells. It is hoped that the slightly modified procedure meets the need for a simple technique to define mononuclear blood cells for clinical purposes.     

In vitro effect of lycopene on cytokine production by human peripheral blood mononuclear cells

There is evidence indicating that regular consumption of tomato products is associated with favorable immunomodulatory effects. In addition, tomato extracts have been shown to possess antioxidant, anticarcinogenic and antithrombotic activity in vitro. Since tomatoes are rich in carotenoids and particularly in lycopene--the pigment responsible for the red color of tomatoes--the present work was designed to examine the in vitro effect of lycopene on cytokine production by peripheral blood mononuclear cells (PBMC) from 15 healthy subjects. First, 2 x 10(6) PBMC suspended in 1 ml of conditioned medium were incubated over a period of 24 and 48 hours without or with the following concentrations of lycopene: 0.25, 0.5, 1.0, 2.0 and 4.0 microM. The production of the subsequent cytokines was evaluated: IL-1beta, IL-1ra, IL-2, IL-6 and IL-10, as well as TNFalpha and IFNgamma. Lycopene induced a dose-dependent increase in IL1beta, and TNFalpha production and a decrease in IL-2, IL-10 and IFNgamma secretion, whereas that of IL-6 and IL-1ra was not affected. It is concluded that understanding the role of lycopene in modulation of the immune system may promote decisions as for dietary supplementation of lycopene for reducing the risk of certain diseases.     

Characterization of bilirubin transport system by human peripheral blood mononuclear cells.

Decreased immune responses have been observed in hyperbilirubinemic patients. This study investigates bilirubin transport into human peripheral blood mononuclear cells (PBMNCs). In vitro incubation of PBMNCs at 37 degrees C with 0-12 mg/dl bilirubin in solution with a fixed bovine serum albumin (BSA) concentration (3.0 g/dl) resulted in a dose-dependent increase of intracellular bilirubin in both monocytes and lymphocytes. Bilirubin uptake in monocytes was significantly higher (up to 2.7 times) than in lymphocytes under the same culture conditions. When PBMNCs were incubated with varying concentrations of bilirubin (0-16 mg/dl) in fixed BSA (3.0 g/dl) solution or at a fixed bilirubin/albumin molar ratio (0.4), the initial velocity of uptake in both cell fractions was proportional to the free (unbound to albumin) bilirubin concentration rather than the total bilirubin concentration. Bilirubin uptake by both cell fractions was significantly inhibited by treatment with metabolic inhibitors. Bilirubin uptake by monocytes continued to increase in parallel with incubation temperature from 0 degrees C to 40 degrees C, whereas uptake by lymphocytes reached a maximal level at 20 degrees C and remained constant thereafter. These results suggest that monocytes and lymphocytes incorporate bilirubin in proportion to the free bilirubin concentration and this function may rely on different energy-dependent mechanisms.     

In vitro production of IgE by human peripheral blood mononuclear cells. I. Rate of IgE biosynthesis

IgE protein and grass-specific IgE antibodies were detected in the supernatants of 7-day cultures of unstimulated and pokeweed mitogen (PWM) stimulated human blood mononuclear cells from non-atopic and grass pollen-sensitive individuals. Significant amounts of IgE protein were detected in culture supernatants of grass-sensitive individuals and, even at lower levels, in those of non-atopic subjects. In contrast, detectable amounts of grass-specific antibodies were found only in the culture supernatants of grass-sensitive subjects. The mean values of total and grass-specific IgE detected in the supernatants of unstimulated and PWM-stimulated cultures did not differ statistically. Time sequence studies showed that IgE concentrations, measured in the 7-day supernatants, were due to a continuous release from the cells of IgE quantities progressively decreasing up to days 7 or 8. Comparison of the IgE protein and IgE antibody found in the 7-day culture supernatants to those released from initial cell pellets by treatment with acid buffer or freezing and thawing, showed that the IgE detected in 7-day supernatants could result, in part, from the release of cytophilic IgE bound to basophil or other cell types and in part also from the release of preformed lymphocyte cytoplasmic IgE into the supernatant fluids during the course of culture. In most non-atopic subjects and in some grass-sensitive patients the preformed IgE accounted virtually for the total IgE detected in the 7-day culture supernatants. However, the increase of IgE above the levels measured in the initial cell pellets, which was found in most grass-sensitive subjects, clearly reflected newly synthesized IgE. Both cycloheximide and puromycin were capable of reducing significantly the IgE concentration in culture supernatants when it was greater than the amount found in the initial cell pellets. The treatment of cells with mitomycin C was also able to decrease significantly the amount of IgE released in the supernatant after day 3 of culture.     

Inhibition of endotoxin-induced interleukin-6 production by synthetic lipid A partial structures in human peripheral blood mononuclear cells.

The effect of two synthetic lipid A partial structures, compound 406 (or LA-14-PP, identical in structure to the lipid A precursor, known as Ia or IVa) and compound 401 (lipid X), on the in vitro modulation of endotoxin (lipopolysaccharide)-induced interleukin-6 production by human blood mononuclear cells was investigated. Lipopolysaccharide of Salmonella abortus equi and synthetic Escherichia coli-type lipid A (compound 506, or LA-15-PP) had potent interleukin-6-inducing capacities. The maximum release of interleukin-6 was found after stimulation with 1 to 10 ng of lipopolysaccharide or 10 to 100 ng of synthetic E. coli-type lipid A per ml. Both synthetic lipid A partial structures (compounds 406 and 401) failed to induce interleukin-6 release. However, they inhibited lipopolysaccharide- or lipid A-induced interleukin-6 production in a dose-dependent manner. Inhibition was found not only in mononuclear cells but also in purified monocytes and was not due to a shift in the kinetics of cytokine production. Suppression was manifested in the early stage of interleukin-6 production. Inhibition was also found in the presence of recombinant gamma interferon, indicating that compound 406 and recombinant gamma interferon act in different, independent pathways. Our data, therefore, indicate that the inhibition of interleukin-6 production by lipid A partial structures may help elucidate the mechanism of interaction of the lipid A component of lipopolysaccharide with immune cells in the inflammatory reaction during gram-negative infection.     

Resveratrol suppresses interferon-gamma-induced biochemical pathways in human peripheral blood mononuclear cells in vitro

A wide range of biological activities of resveratrol (3,5,4′-trihydroxystilbene) in vitro and in vivo has been proved, including antioxidant, antitumor, and also anti-inflammatory effects. Resveratrol found in, e.g., grapes and red wine has been suggested to counteract the progression of coronary heart disease by lowering serum lipid concentrations and inhibiting platelet aggregation. Cellular immune activation is known to be involved crucially in the pathogenesis of coronary heart diseases. In this in vitro study, the modulatory effect of resveratrol on two interferon-γ-mediated pathways, the degradation of tryptophan by the enzyme indoleamine 2,3-dioxygenase, and the production of neopterin by activation of the GTP-cyclohydrolase I, was tested. Cultures of human peripheral blood mononuclear cells were exposed to resveratrol, in combination with mitogenic stimulation. A significant down-regulatory effect of resveratrol on both biochemical pathways was found, and also the production of Th1-type cytokine interferon-γ was significantly suppressed. If these results can be verified in vivo, an explanation is provided how resveratrol may interfere with immune activation and cytokine cascades, which are important in the development and progression of cardiovascular disorders and also other diseases.     

Aspirin down-regulates tryptophan degradation in stimulated human peripheral blood mononuclear cells in vitro

Acetylsalicylic acid (aspirin) is one of the most widely used drugs worldwide, due mainly to its broad therapeutic spectrum with anti-inflammatory, antipyretic, antithrombotic and analgesic effects. However, the exact mechanisms by which aspirin influences inflammation, pain and immune system activation are only partly understood. Within activation of the cellular immune system, Th1-type cytokine interferon (IFN)-gamma induces enzyme indoleamine-2,3-dioxygenase (IDO) which converts tryptophan to kynurenine. In parallel, IFN-gamma induces enzyme GTP-cyclohydrolase I, which gives rise to neopterin production by activated human macrophages. Similarly, tryptophan degradation and neopterin formation increase during several disease states involving Th1-type immune activation. Using stimulated human peripheral blood mononuclear cells (PBMC), the effect of aspirin on tryptophan degradation and neopterin production was investigated. Stimulation of PBMC with mitogens concanavalin A, phytohaemagglutinin and pokeweed mitogen induced significant tryptophan catabolism as was reflected by a decline in tryptophan levels and a parallel increase in kynurenine concentrations compared with unstimulated cells. In parallel, neopterin production was enhanced. Treatment of stimulated PBMC with increasing doses of 1-5 mM aspirin significantly decreased stimulation-induced tryptophan degradation and neopterin production as well. All the effects of aspirin were dose-dependent. The parallel influence of aspirin on both biochemical pathways implies that there was no direct inhibitory effect of aspirin on IDO; rather, it inhibits production of IFN-gamma in mitogen-treated PBMC. The influence of aspirin on biochemical pathways induced by IFN-gamma may represent an important part of its broad pharmacological effect.     

Replication of rubella virus in human mononuclear blood cells.

Rubella virus was capable of replicating in both unstimulated and phytohemagglutinin-stimulated cultures of human mononuclear blood cells. Monocyte-derived macrophages were the main cell type responsible for viral replication. The susceptibility of macrophages increased during cultivation. Phytohemagglutinin-stimulated lymphocytes were able to support replication to a limited degree. No viral replication was detected in unstimulated lymphocytes. Both stimulation and viral replication in phytohemagglutinin-treated lymphocyte cultures were enhanced by the addition of murine macrophages. Human leukocyte interferon depressed the production of virus in these combined cultures. The finding that rubella virus is able to replicate in human lymphocytes as well as in macrophages may contribute to understanding the mechanisms of the suppressive effect of the virus on in vitro lymphocyte phytohemagglutinin responsiveness and in vivo immune functions.