Differential modulation of interleukin-4 and interleukin-13 secretion from human peripheral blood mononuclear cells.

Interleukin-4 (IL-4) and IL-13 which have been implicated in the pathogenesis of atopic reactions elicit many of the same biologic responses. Therefore, time- and stimulus-dependent differences in the regulation of IL-4 and IL-13 production could be of relevance to their biological effects. In this study we tested the hypothesis that stimulation of peripheral blood mononuclear cells (PBMCs) with different inducers of cell activation would result in a differential expression of IL-4 and IL-13. For this purpose, PBMCs of nonatopic volunteers were incubated with phytohaemagglutinin (PHA), phorbolester (PMA), calcium ionophore A23187, or IL-3. The effect of these stimuli on IL-4 and IL-13 production were analysed by enzyme-linked immunoassay (ELISA) in supernatants of cultured PBMCs. Incubation of PBMCs with A23167 and PHA induced both a dose- and time-dependent increase in IL-4 and IL-13 release. A23187 induced concentrations of IL-4 were higher than those of IL-13 whereas IL-4 release following stimulation with PHA was considerably higher for IL-13 compared to IL-4. In contrast, there was a selective increase in IL-13 but not IL-4 concentrations following stimulation of PBMCs with PMA and IL-3 in vitro. In conclusion in this study evidence is provided that IL-4 and IL-13 production are regulated differently which might explain their functional redundancy.     

Vitamin C and E suppress mitogen-stimulated peripheral blood mononuclear cells in vitro

BACKGROUND/AIMS: The antioxidant properties of vitamin C and E are considered to be important for their anti-inflammatory activity. Recently, antioxidant resveratrol was found to suppress neopterin production and tryptophan degradation in mitogen-treated human peripheral blood mononuclear cells. METHODS: In this study, the effects of vitamin C and E were investigated in unstimulated peripheral blood mononuclear cells and in cells stimulated with the mitogens phytohaemagglutinin and concanavalin A in vitro. RESULTS: The mitogens induced a significant production of neopterin and a degradation of tryptophan. Vitamin C (0.1-10 microM) and E (5-100 microM) suppressed these immunobiological pathways in a dose-dependent way (p < 0.01). CONCLUSION: Neopterin production and tryptophan degradation in monocyte-derived macrophages are both triggered by the pro-inflammatory cytokine interferon-gamma. Thus, their concurrent suppression by vitamin C and E suggests an effect on the formation and release of this cytokine by stimulated T cells. These findings may be related to the general health benefits which are associated with the antioxidant nature of these vitamins.     

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.     

The detection by immunofluorescence of distinct cell populations producing interleukin-1 alpha and interleukin-1 beta in activated human peripheral blood

An immunofluorescent staining method using specific monoclonal antibodies was used to detect IL-1 alpha and IL-1 beta in individual cells in stimulated human peripheral blood. No staining was seen in unstimulated cells but intense, maximal staining of approximately 5% of the cells was seen 20-24 h after activation with PHA/PMA. The large irregularly shaped stained cells were surrounded by smaller unstained cells with lymphocyte-like morphology. By 44 h post activation a few cells only showed weak staining. The staining pattern was different for the two molecules studied, with a granular pattern for IL-1 alpha staining and diffuse cytoplasmic staining for IL-1 beta. Staining post-activation could be abolished by preincubation of the monoclonal antibody with the appropriate recombinant IL-1, but not by pre-incubation with the other IL-1 type. When both anti-IL-1 alpha and anti-IL-1 beta were used together two populations of cells were identified; one had the granular staining as seen with anti-IL-1 alpha alone and the other had the diffuse staining pattern as seen with anti-IL-1 beta. The percentage of cells showing bright staining with anti-IL-1 alpha and anti-IL-1 beta together was approximately equal to the sum of the percentage of cells staining for IL-1 alpha or IL-1 beta alone. This study demonstrates a method for the detection of individual IL-1 alpha- and IL-1 beta- producing cells and suggests that in activated human peripheral blood IL-1 alpha and IL-1 beta are produced by separate populations of cells.     

In vitro modulation of keratinocyte-derived interleukin-1 alpha (IL-1 alpha) and peripheral blood mononuclear cell-derived IL-1 beta release in response to cutaneous commensal microorganisms.

The ability of a range of skin commensal microorganisms to modulate interleukin-1 (IL-1) release by cultured human keratinocytes and peripheral blood mononuclear cells (PBMCs) was investigated by a combination of enzyme-linked immunosorbent assays and bioassays. Three fractions (formaldehyde-treated whole cells, culture supernatants, and cellular fractions) were prepared from Propionibacterium acnes, Propionibacterium granulosum, Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus hominis, and Malassezia furfur serovar B. The levels of immunochemical IL-1 alpha released by cultured keratinocytes during coincubations with these microbial fractions ranged from 0 to 136 pg/ml and were maximal after 72 h. No microbial fraction consistently upregulated immunochemical IL-1 alpha release by freshly isolated keratinocytes from two donors and a transformed cell line, all of which produced the cytokine constitutively to various extents. Bioassays revealed that most of the IL-1 released was biologically inactive. In contrast, whole cells of formaldehyde-treated P. granulosum and S. epidermidis significantly stimulated release of IL-1 beta by PBMCs from three donors compared with the negative control (culture medium). Release was maximal at 24 h. Coincubation with intact cells of the yeast M. furfur significantly decreased levels of IL-1 beta below the values for the negative control by PBMCs from all three donors. There was good correlation between bioassay data and immunoassay data for IL-1 beta, and the depressive effect of M. furfur cells on cytokine production by all three cultures of PBMCs was mirrored in the levels of bioactive cytokine. This reduction in IL-1 beta release by PBMCs by M. furfur may provide an explanation why dermatoses thought to be caused by this yeast are essentially noninflammatory or only mildly inflammatory.     

杂色曲霉素对体外培养HPBMcIFN-γ分泌的影响

目的:探讨杂色曲霉素(ST)对人T淋巴细胞分泌功能的影响。方法:采用双抗体夹心ELISA方法对ST作用后人外周血单核细胞(HPBMc)培养上清液中γ-干扰素(IFN-γ)的分泌水平进行检测。结果:不同浓度ST对IFN-γ分泌的影响不同,较低浓度ST0.03125mg/L-0.12500mg/L对IFN-γ的分泌有一定抑制作用(P＜0.05),而在0.25mg/L以上浓度ST可刺激IFN-γ分泌,至1mg/L达到高峰(P＜0.05)。在0.25mg/L-1.00mg/L的浓度范围内,ST浓度和IFN-γ分泌呈正相关(r=0.492,P＜0.01);在ST1mg/L作用1h-64h的时间范围内,ST对IFN-γ分泌的影响依时间不同作用也不同,在4h-8hIFN-γ的分泌降低(8h,P＜0.05),而以后各组随着ST作用时间的延长IFN-γ水平逐渐增高,尤以32h组增高最为明显(P＜0.05),在ST作用16h-64h的时间范围内,IFN-γ的分泌随着ST作用时间的延长而逐渐增加,两者呈正相关(r=0.736,P＜0.01)。结论:ST对HPBMcIFN-γ分泌的影响表现在较低浓度和较短时间抑制IFN-γ分泌,而较高浓度和较长时间则诱导IFN-γ分泌,但继续增加ST浓度和作用时间并不继续增加IFN-γ分泌。     

Acellular components of Chlamydia pneumoniae stimulate cytokine production in human blood mononuclear cells

Accumulating evidence suggest that infection with Chlamydia pneumoniae is associated with atherosclerosis, but the mechanisms involved remain unclear. Inflammation is important in the initial phase of atherogenesis, and cytokines are important in the initiation and progression of inflammation. The aim of this study was to assess the capacity of acellular components of C. pneumoniae to stimulate the production of pro-inflammatory cytokines and chemokines. Peripheral blood mononuclear cells were stimulated in vitro with sonicated C. pneumoniae. Significant amounts of TNF-alpha, IL-1, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) were produced. Inhibition of endotoxin using polymyxin B revealed that chlamydial endotoxin plays a minor role in the cytokine induction. Neutralization of TNF by TNF-binding protein and blockade of IL-1 receptors by IL-1 receptor antagonist revealed that TNF, IL-1 and IL-6 production was independent from each other, whereas IL-8 synthesis was strongly dependent on endogenous TNF and IL-1. In contrast, synthesis of MCP-1 and MIP-1alpha was dependent on endogenous TNF, but not IL-1. In conclusion, acellular components of C. pneumoniae are a potent stimulus for cytokine production, and this mechanism may have an important role in the inflammatory aspects of atherogenesis.     

Simvastatin induces interleukin-18 production in human peripheral blood mononuclear cells

The effects of statins on immune response depend on the inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase and leukocyte function-associated antigen (LFA)-1, which is a ligand of intercellular adhesion molecule (ICAM)-1. Simvastatin, an HMG-CoA reductase inhibitor with mild inhibition of LFA-1, induced the production of interleukin (IL)-18, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in human peripheral blood mononuclear cells (PBMC). The IL-18 production is located upstream of the cytokine cascade activated by simvastatin. Moreover, simvastatin concentration-dependently inhibited the expression of ICAM-1 and induced the expression of CD40 on monocytes. In the presence of IL-18, simvastatin suppressed the expression of ICAM-1 and CD40 as well as the production of IL-12, TNF-alpha and IFN-gamma in PBMC, contributing to the anti-inflammatory effect of simvastatin. The effects of simvastatin were abolished by the addition of the product of the HMG-CoA reductase, mevalonate, indicating the involvement of HMG-CoA reductase in the action of simvastatin.     

Regulation of interleukin-2 induced interleukin-5 and interleukin-13 production in human peripheral blood mononuclear cells

Adjuvant interleukin (IL)-2 immunotherapy has been used for many years for a variety of malignant and nonmalignant entities. In many cases, a dose escalation might have seemed desirable, but was prevented by the rather severe adverse effects of systemic IL-2 application. Only recently has the regulation of IL-2 induced cytotoxicity been understood better, so that now efforts can be aimed at the design of cytokine cocktails that would selectively induce cytotoxicity but result in as few adverse effects as possible. Previously, induction of IL-5 under systemic IL-2 therapy has been described, and a number of the side-effects have been attributed to this event. We therefore investigated the regulation of IL-2 induced production of IL-5 and IL-13 (which, similarly to IL-5, is a mediator of allergy-like symptoms). At the same time, the effects of regulatory cytokines, such as IL-4, IL-10 and IL-12, on interferon-gamma (IFN-gamma), the major cytotoxic mediator of IL-2 therapy, were studied. All three have been discussed as antitumour immunotherapeutics, either alone or in combination with IL-2. In anti-CD3-treated peripheral blood mononuclear cells, IL-2 induced IL-5 and IL-13 alongside IFN-gamma, IL-10 and IL-12. In the presence of IL-2, inhibition of endogenous IL-12 production further enhanced the IL-5 and IL-13 responses, while IFN-gamma and IL-10 were markedly suppressed. Co-incubation with IL-2 and IL-12 suppressed IL-5/IL-13 below, but enhanced IFN-gamma and IL-10 above, levels induced by IL-2 alone. IL-10 was suppressive on all the investigated cytokines, while IL-4 interfered with IL-2 induced IFN-gamma and IL-12 production, but was additive to IL-2 in its effect on IL-5 and IL-13. These data suggest that the combination of IL-12 with IL-2 would enhance the cytotoxic activity of this regimen, but might reduce its adverse effects.     

Analysis of the spontaneous in vitro anti-HIV-1 antibody secretion by peripheral blood mononuclear cells in HIV-1 infection.

We studied the spontaneous in vitro secretion of anti-HIV-1 antibodies by peripheral blood mononuclear cells (PBMC) from HIV-1-infected patients. Specific antibody production was detected in supernatants of PBMC cultures using an ELISA; HIV-1 specificity was confirmed by antigen adsorption and Western blotting. This antibody secretion was found to be an active phenomenon and was not due to a release of plasma antibodies passively adsorbed onto the cell membranes. In all positive supernatants, anti-HIV-1-secreted antibodies were directed against env-encoded antigens and many supernatants also contained antibodies to pol- and gag-encoded antigens. PBMC from all HIV-1-infected patients tested (140 adults and 18 infants) secreted anti-HIV-1 antibodies. This production was found during all the clinical stages of HIV-1 infection. Our results suggest that this spontaneous HIV-1-specific antibody secretion represents a marker of HIV-1 infection. Detection of these antibodies could be a valuable tool for early confirmation of HIV-1 infection in neonates born to HIV-1-seropositive mothers.     

IgG aggregates of different sizes stimulate or suppress Ig secretion by human lymphocytesin vitro

The regulatory effects of IgG aggregates on Ig productionin vitro by human peripheral blood lymphocytes were shown to be highly dependent on the aggregate size and the degree of mitogenic stimulation. Covalently linked oligometers of IgG were prepared with dimethylsuberimidate cross-linking and chromatographic separation; larger aggregates were prepared by heating (63°C) and preparative zonal ultracentrifugation. The storage and culture conditions used were shown to preserve the stability of aggregate sizes. Although both positive and negative regulatory effects were seen with cells isolated directly from blood, more predictable dose-related effects were seen if cells were vigorously washed, possibly due to the removal of IgG or natural immune complexes bound by the cellsin vivo. Some preparations of small IgG oligomers produced marked stimulation of Ig production, especially in cells cultured without mitogen or with suboptimal pokeweed mitogen doses. Aggregates containing six or more IgGs suppressed Ig production, especially when cells were stimulated by mitogen at optimal concentrations.     

Influence of dental amalgam and heavy metal cations on in vitro interleukin-1beta production by human peripheral blood mononuclear cells

The influence of dental amalgam and heavy metal cations on interleukin-1beta (IL-1beta) expression by peripheral blood mononuclear cells from healthy donors was studied. A marked decrease in the production of IL-1beta was caused by freshly prepared amalgam or amalgam-conditioned culture medium, but not by amalgam aged for 6 weeks. When metal cations were added as salts, Cu(2+), Hg(2+), and Ag(+) at high concentrations (33.3 and 333.3 microM) were highly inhibitory. Among other heavy metal cations, Au(3+), Pt(4+), Ni(2+), Pd(2+), but not Ga(3+) or Sn(2+), inhibited IL-1beta production in a concentration-dependent manner. Flow cytometry studies indicated that Hg(2+) and Ag(+) strongly reduced the percentage of CD14(+) cells containing IL-1beta intracellularly. As shown by Northern blot analysis, Hg(2+) inhibited the level of IL-1beta-specific mRNA by 28% at 3.3 microM and completely at 33.3 microM. Only slight inhibitory effects were induced by Cu(2+) at 33.3 microM. Interestingly, Ag(+) at a concentration of 3.3 microM increased twofold the amount of IL-1beta-specific mRNA. Our data show that IL-1beta production is altered at protein and mRNA levels by components released from fresh amalgam and by other heavy metal cations, suggesting a role of these cations in changes in the cell phenotype and IL-1-mediated cell functions.     

In vitro response of peripheral blood mononuclear cells to phytohemagglutinin and interleukin-2

The serological determination of class II antigens is still a mandatory test prior to allotransplantation. It is known that these antigens are normally expressed on B lymphocytes and monocytes. The B lymphocytes that constitute 10% to 15% of total blood lymphocytes are the cells currently used for HLA-DR typing. To avoid HLA-DR typing difficulties, or even impossibilities that are frequently encountered among some patient groups, we studied the response of peripheral blood mononuclear cells--as an alternative source of cells for class II antigen typing--to in vitro mitogen and interleukin-2 activation and propagation. Although the patients included in this study were selected having previously known HLA-DR typing difficulties, all could be adequately typed by this method.     

Non-peptidic delta-opioid receptor antagonists suppress mitogen-induced tryptophan degradation in peripheral blood mononuclear cells in vitro

Opioid receptors are expressed not only on neuroendocrine cells but also on immunocompetent cells such as lymphocytes, monocytes and macrophages. micro-Opioid receptor agonists were found to exert immunosuppressive effects, whereas delta-opioid receptor agonists have been shown to act as immunostimulants. delta-Opioid receptor agonists stimulate T and B cells and activate granulocytes and monocytes, conversely, immunostimulation can be blocked by the non-peptidic delta-opioid receptor antagonist (NTI). We investigated the impact of NTI and of the two structurally related compounds HS-378 and HS-459 on degradation of tryptophan and formation of neopterin in mitogen-stimulated human peripheral blood mononuclear cells (PBMC). Both these biochemical pathways were found to be suppressed by all three opioid receptor antagonists, HS-378 and HS-459 exhibiting slightly greater potency than NTI. The suppression of tryptophan degradation suggests that the tested delta-opioid antagonists are able to influence the serotonergic system via a non-opioid action.     

Interleukin-4 downregulates interleukin-6 production in human peripheral blood mononuclear cells

We report that recombinant human interleukin-4 (IL-4) downregulates interleukin-6 (IL-6) production by human peripheral blood mononuclear cells (PBMC). PBMC were preincubated for up to 24 hr in the presence of IL-4 (100 U/ml) and then activated with lipopolysaccharide B Escherichia coli 026:B6 (LPS, 10 micrograms/ml), recombinant human tumor necrosis factor-alpha (TNF-alpha, 200 U/ml), or Concanavalin A (Con A, 10 micrograms/ml). Although all these signals induced IL-6 production, IL-4-treated cells produced significantly reduced levels of IL-6 protein. This effect was dose and time dependent. We conclude that IL-4 is a potent downregulatory modulator of IL-6 expression in human PBMC.     

Inhibition of varicella-zoster virus in vitro by human peripheral blood mononuclear cells.

A functional in vitro assay of cell-mediated immunity to varicella-zoster virus (VZV) is described. This procedure uses an enzyme-linked immunosorbent assay (ELISA) to measure the inhibitory effect of human peripheral blood mononuclear cells on VZV antigen production by VZV-infected cell monolayers. When mononuclear cells from VZV-immune, tetanus-immune donors were stimulated with either VZV antigen or tetanus toxoid they reduced VZV antigen production. In contrast, mononuclear cells from VZV-nonimmune, tetanus-immune donors reduced VZV antigen only when stimulated with tetanus toxoid, but not when stimulated with VZV antigen. Cell-free supernatants recovered from the VZV inhibition assays contained the anti-VZV activity. The magnitude of the anti-VZV activity of the supernatants equalled the inhibition observed when the stimulated mononuclear cells were added to the VZV-infected monolayers. Treatment of either mononuclear cells or supernatants with anti-interferon gamma antibody indicated that their VZV inhibitory capability was largely due to the production of interferon gamma by stimulated mononuclear cells.     

Atorvastatin suppresses interferon-gamma -induced neopterin formation and tryptophan degradation in human peripheral blood mononuclear cells and in monocytic cell lines

Inhibitors of 3-hydroxy-3methylglutaryl-co-enzyme A (HMG-CoA) reductase, so-called statins, are used in medical practice because of their lipid-lowering effect and to reduce the risk of coronary heart disease. Recent findings indicate that statins also have anti-inflammatory properties and can modulate the immune response. In vitro, we investigated the effect of atorvastatin on the T cell/macrophage system in peripheral blood mononuclear cells (PBMC) and in the human monocytic cell lines THP-1 and MonoMac6. We monitored neopterin production and tryptophan degradation in PBMC after treatment with 10 micro m and 100 micro m atorvastatin in the presence or absence of 100 U/ml IFN-gamma, 10 micro g/ml phytohaemagglutinin (PHA) or 10 micro g/ml concanavalin A (ConA) and in monocytic cell lines THP-1 and MonoMac6 with or without stimulation with 100 U/ml IFN-gamma or 10 ng/ml to 1 micro g/ml lipopolysaccharide (LPS). In stimulated PBMC 100 micro m atorvastatin inhibited neopterin formation and tryptophan degradation completely, whereas 10 micro m atorvastatin was only partially effective. Also in monocytic cell lines THP-1 and MonoMac6, atorvastatin was able to suppress IFN-gamma- and LPS-induced formation of neopterin and degradation of tryptophan. Our data from PBMC agree well with previous investigations that statins inhibit T cell activation within the cellular immune response. In addition we demonstrate that atorvastatin directly inhibits IFN-gamma-mediated pathways in monocytic cells, suggesting that both immunoreactivity of T cells and of monocyte-derived macrophages are down-regulated by this statin.     

Spontaneous secretion of interleukin-4, interleukin-10 and interferon-gamma by first trimester decidual mononuclear cells

PROBLEM: A T-helper cell type 2 (Th2) cytokine dominated microenvironment has been predicted to be crucial for successful pregnancy. However, little information is available about local cytokine secretion in the human decidua. We determined the spontaneous secretion of interleukin-4 (IL-4), interferon-gamma (IFN-gamma) and IL-10 by decidual mononuclear cells at the single cell level and compared it with their secretion by peripheral blood mononuclear cells (PBMC) in the first trimester of pregnancy. METHODS OF STUDY: The cytokine secretion from decidual and blood cells was detected by a sensitive enzyme-linked immunosorbent spot-forming cell (ELISPOT)-assay. RESULTS: Cells secreting IL-4 (median 153, range 8-530), IL-10 (median 188, range 32-1600) and IFN-gamma (median 123, range 15-1140) were detected in all decidual and blood samples. The cytokine secretion showed a co-linear pattern in both the blood and decidua, i.e. when one cytokine was secreted at high levels, the others followed the trend. No correlation was found between the number of cytokine secreting cells in blood and decidua for any of the cytokines. CONCLUSIONS: Interleukin-4 and IL-10 are locally secreted in the decidua early during normal pregnancy, probably counteracting the fetal rejecting effects of co-expressed IFN-gamma. The cytokine secretion by blood cells does not generally reflect the local secretion pattern during first trimester pregnancy.