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.     

In vitro effect of statins on cytokine production and mitogen response of human peripheral blood mononuclear cells

The in vitro effect of the hydrophilic statin - pravastatin - and three hydrophobic statins - atorvastatin, lovastatin and simvastatin - on the production of IL-1beta, IL-1ra, IL-2, IL-6 and IFN-gamma by human peripheral blood mononuclear cells (PBMC) and their response to mitogens was examined. Lovastatin and simvastatin increased the production of IL-1beta in a dose dependent manner and reduced secretion of IL-1ra at high concentration. These two statins exerted a dose dependent inhibitory effect on IL-2 production and reduced the secretion of IFNgamma at high dose. Atorvastatin did not affect IL-1beta, but suppressed IL-1ra, IL-2 and IFNgamma production. Atorvastatin, lovastatin and simvastatin caused a dose dependent inhibition of mitogen-induced proliferation of PBMC. IL-6 production was not affected by any one of the statins. While pravastatin caused a slight reduction in the proliferative response of PBMC to PHA, it did not affect either their response to Con A and PWM, or the secretion of cytokines tested.     

In vitro modulation of proliferation and cytokine production by human peripheral blood mononuclear cells from subjects with various forms of coccidioidomycosis.

Using peripheral blood mononuclear cells (PBMC) from individuals with or without coccidioidal delayed-type hypersensitivity (DTH), we examined and attempted to modulate the in vitro responses of PBMC from various donors to the coccidioidal antigen toluene spherule lysate (TSL). Among healthy DTH-positive donors, 100 ng of human recombinant interleukin-10 (IL-10) per ml suppressed both PBMC proliferation (P = 0.01) and gamma interferon (IFN-gamma) and IL-12 production (for both, P < 0.05). In vitro proliferation and production of IFN-gamma and IL-12 by PBMC were significantly higher in DTH-positive donors with active coccidioidomycosis than in healthy, nonimmune controls (P < 0.05) but not in active DTH-negative donors with or without human immunodeficiency virus infection (for both, P > 0.05). Human recombinant IL-12 increased IFN-gamma production by PBMC from active, DTH-positive donors (P = 0.01) but not by PBMC from DTH-negative groups. For healthy DTH-positive donors, the median antigen-reactive cell frequency per 10(5) PBMC was 3.7, compared to 1.7 in DTH-negative donors with active coccidioidomycosis (P = 0.03). These data indicate that the in vitro TSL response is highly dependent on coccidioidal DTH. Not only do PBMC from individuals with DTH appear to respond to TSL, but their response can be modulated in vitro with either IL-10 or IL-12. On the other hand, PBMC from DTH-negative individuals do not respond in vitro to TSL and their response is not modulable, suggesting a lack of antigen response.     

In vitro production of cytokines by peripheral blood mononuclear cells from hydatid patients.

The role of cytokines in human hydatidosis (Echinococcus granulosus infection) was evaluated in immunoassays determining production of IL-4, IL-10 and interferon-gamma (IFN-gamma) in peripheral blood mononuclear cell (PBMC) cultures from 30 hydatid patients and 14 uninfected controls. In cell cultures from hydatid patients parasite and non-parasite antigen stimulation significantly increased IL-4 production (P < or 0.005). Spontaneous and mitogen-driven IL-4 production was similar in patients and controls. IL-10 and IFN-gamma production did not differ statistically in the two groups, even though some hydatid patients produced these cytokines in large amounts. Notably, antigen-driven IFN-gamma concentrations were invariably higher in patients than in uninfected controls. Data analysis showed a relationship between IgE and IgG4 responses and parasite-driven cytokine production. High IgE and IgG4 responders produced high IL-4 and IL-10 concentrations. High IgE responders showed decreased IFN-gamma production, but high IgG4 responders had IFN-gamma levels slightly higher than those of low responders. Cytokine response patterns did not relate to the clinical stage of disease. The significantly increased IL-4 and the high IL-10 concentrations found in PBMC from many hydatid patients in this study are consistent with Th2 cell activation in human hydatidosis. The presence of antigen-driven IFN-gamma production in patients with E. granulosus infection implies concurrent intervention of the Th1 or Th0 cell subset.     

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.     

In vitro IgE production by interleukin 4-stimulated human peripheral blood mononuclear cells is suppressed by rapamycin

Rapamycin (RAPA) is a new immunosuppressant which is 50-fold to 100-fold more potent than cyclosporin A (CyA) in inhibiting cellular immune responses and allograft rejection in animal models of organ transplantation. The drug's effect on in vitro IgE synthesis by interleukin (IL)4-stimulated human peripheral blood mononuclear cells was examined and compared with CyA's effect in this study. RAPA was found to be about 100-fold more potent than CyA in inhibiting IgE synthesis. Its inhibitory effect on IgE production was significant if it was added to the culture before Day 6 of a 14-day culture. The suppression was accompanied by the inhibitory effect on cell proliferation and on IgE-binding factor (IgE-BF) production. IL2 was able to partially reverse CyA- but not RAPA-induced inhibition of IgE production. Commercial B cell growth factor (cBCGF) was not able to reverse either RAPA- or CyA-induced suppression of IgE synthesis. The strong inhibitory effect of RAPA in IgE synthesis may be useful in certain clinical applications where overproduction of pathogenic IgE is a key issue. RAPA can also be used as a tool to dissect the regulation of IgE production.     

In vitro production of IgE-binding factors by human mononuclear cells.

This study documents the production of IgE-binding factors (IgE-BFs) by unstimulated and by mitogen-activated human mononuclear cells. IgE-BFs were detected by a sensitive radioimmunoassay employing monoclonal antibodies to lymphocyte Fc epsilon R (MabER). IgE-BFs were found in the 24-hr CSN of unfractionated tonsillar lymphocytes and of their B-cell but not of their T-cell enriched fractions. When cultured for 1 week, PBMC spontaneously synthesized and released IgE-BFs in the CSN; this was significantly reduced by IgE (10 micrograms/ml). PWM, PHA and Con A significantly increased the production of IgE-BFs by PBMC, and this was not influenced by IgE. The production of IgE-BFs in response to mitogens required interactions between T and non-T cells, and IgE-BFs seemed to be derived mainly from non-T cells. However, low levels of IgE-BFs could be detected in the CSN of highly purified T cells cultured for 1 week in the presence of PHA. The production of IgE-BFs by non-T cells was T-cell dependent and it was mediated by soluble factors released from mitogen-activated T cells. T-cell factors increased the secretion of IgE-BFs by: the macrophage cell line U937, adherent cells, and adherent cell-depleted B-cell preparations. It is concluded that the majority of IgE-BFs produced by cultured human mononuclear cells are derived from B cells and monocytes, and that their production is regulated by T lymphocytes.     

In vitro production of IgE by human peripheral blood mononuclear cells. II. Cells involved in the spontaneous IgE production in atopic patients

Spontaneous IgE production in vitro was investigated in 7-day cultures of unfractionated mononuclear cells (MNC) and MNC subpopulations from atopic patients. Depletion of either phagocytic or adherent cells decreased the amount of IgE detectable in 7-day culture supernatants, but this decrease was due, at least in part, to a loss of cytophilic IgE. Depletion of immunoglobulin-bearing cells (SIg⁺) reduced significantly but did not abolish the spontaneous IgE production in vitro. On the other hand, depletion of IgM-bearing lymphocytes (SIgM⁺), which virtually abolished the production of immunoglobulins of the IgM class, did not change significantly the spontaneous production of IgE. Similarly, no change in the spontaneous production of IgE was found when lymphocyte suspensions were depleted of complement receptor-bearing cells (CR⁺). In contrast, spontaneous IgE production was significantly increased by depletion of T lymphocytes and this increase did not simply reflect the enrichment for IgE-producing cells caused by the fractionation procedure. No significant change in the spontaneous IgE production was found when small numbers of autologous T lymphocytes were added to B cell fractions, whereas the addition of higher concentrations of autologous T cells induced a marked inhibition of the spontaneous IgE production. On the other hand, the addition in culture of pokeweed mitogen (PWM) resulted in a marked reduction of the spontaneous IgE production by B cells, also in the presence of small concentrations of autologous T lymphocytes. Normal T cells were consistently effective in inducing a partial inhibition of the spontaneous IgE production by B cells from atopic patients, whereas T cells from a noticeable proportion of atopic patients were not. These data suggest that MNC responsible for the spontaneous IgE production in atopic subjects are SIgM- and CR-deficient well-differentiated lymphocytes which probably represent the result of an activation which has occurred in vivo. However, this spontaneous IgE production can still be influenced by in vitro manipulation, such as variations in T–B cell ratios or addition of PWM. The results here reported also indicate that normal T cells are generally more effective than T cells from atopic patients in regulating the activity of spontaneous IgE-producing cells present in the blood of atopic subjects.     

In vitro production of IgE by human peripheral blood mononuclear cells. IV. Modulation by allergen of the spontaneous IgE antibody biosynthesis.

Peripheral blood lymphocytes (PBL) from a proportion of grass-sensitive patients, studied during or immediately after the grass pollination period, showed spontaneous production in vitro of grass-specific IgE antibody, whereas PBL from atopic patients sensitive to allergens other than grass pollens or non-atopic individuals did not. Pre-incubation of IgE antibody producing PBL from grass-sensitive patients with minute amounts of a mixed grass pollen (MGP) extract or Rye grass antigen Group I (Rye I) usually resulted in a reduction of the spontaneous production in vitro of IgE protein and in a marked inhibition of the spontaneous production in vitro of grass-specific IgE antibody. This antigen-specific inhibition was not mediated by T lymphocytes, but it was apparently due to a signal directly delivered by antigen to the spontaneously IgE antibody producing cells. The results support the concept that the activity of cells responsible for the persistent IgE antibody formation in vitro in atopic patients can be modulated by antigen.     

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.     

In vitro cytokine production by mononuclear cells exposed to bone cement extracts

The authors evaluated the ability of bone cement to modify the profile of pro-inflammatory cytokines secreted by the immune cells. Peripheral blood mononuclear cells (PBMC) collected from healthy individuals were cultured with cement extracts and tested to assess the release of IL-1beta, TNFalpha, GM-CSF and IL-6 in both unstimulated and PHA-stimulated PBMC. The cytokine release of unstimulated PBMC was very poor, and in particular the IL-1beta was undetectable: the addition of cement extract increased both TNFalpha and GM-CSF release and decreased IL-6, sometimes significantly. The most recurrent observation in PHA-stimulated PBMCs exposed to bone cement extract was the increase in both IL-1beta and IL-6 release, while both the mean concentration and the index of release of TNFalpha and GM-CSF were changeable. In conclusion our results showed that leachable components of some bone cements can induce in vitro the release of pro-inflammatory cytokines which are known to be involved in the bone resorption associated with aseptic loosening of hip prostheses. These findings allowed us to identify materials endowed with the highest inflammatory power.     

Testosterone inhibits immunoglobulin production by human peripheral blood mononuclear cells

We studied the in vitro effect of testosterone on spontaneous immunoglobulin production by human peripheral blood mononuclear cells (PBMC). Testosterone inhibited IgG and IgM production by PBMC both from males and females. The inhibitory effect of testosterone was revealed at doses more than 1 nm, increased dose-dependently, and reached a plateau at 100 nm. At doses <1000 nm, testosterone did not reduce cell viability. Testosterone treatment reduced IgG production by 59.0% and that of IgM by 61.3% compared with control. Immunoglobulin production by B cells was also suppressed by testosterone, though the magnitude of the suppressive effect on B cells was lower than that on whole PBMC; testosterone-induced decrease of IgG production compared with control was 26.9% and that of IgM was 24.9%. Exogenous IL-6 partially restored the impaired immunoglobulin production of testosterone-treated PBMC; IgG production in testosterone culture was increased by IL-6 from 35.6% to 66.5% of control and that of IgM was also increased from 38.9% to 71.2%, respectively. Testosterone treatment reduced IL-6 production of monocytes by 78.4% compared with control, but neither affected that of T cells or B cells. These results suggest that testosterone may suppress immunoglobulin production of human PBMC directly by inhibiting B cell activity and indirectly by reducing IL-6 production of monocytes. It is thus indicated that this hormone may have protective and therapeutic effects on human autoimmune diseases.     

Immune complex size and complement regulate cytokine production by peripheral blood mononuclear cells

We have previously shown that immune complexes isolated from children with juvenile rheumatoid arthritis are heterogeneous in their size, composition, and proinflammatory capacities. The experiments described here were undertaken to clarify further the roles of size and composition in determining the proinflammatory effects of immune complexes. We incubated peripheral blood mononuclear cells (PBMCs) with different soluble immune complex preparations: opsonized complexes, which were formed in the presence of serum, unopsonized complexes, which were formed in the absence of serum, and immune precipitates solubilized by complement after their formation. ELISA assays showed that immune complexes formed in the presence of complement were less efficient than unopsonized complexes in inducing IL-1beta and IL-8 secretion from leukocytes. Solubilized immune precipitates showed intermediate capacity to stimulate the release of both cytokines. Complexes formed in heat-inactivated serum were as efficient as unopsonized complexes in eliciting cytokine secretion from the cells. The capacity of complement to regulate cytokine secretion from leukocytes was related, at least in part, to immune complex size. Sucrose density gradients showed unopsonized complexes and solubilized immune precipitates were larger than opsonized immune complexes. In contrast, fluid-phase binding of C4 to immune complexes, which did not appreciably change immune complex size, substantially increased IL-1beta secretion from 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.     

Determinants of cytokine induction by small interfering RNA in human peripheral blood mononuclear cells.

Synthetic small interfering RNAs (siRNAs) can trigger a strong innate immune response in mammalian cells. This nonspecific side effect may hinder the application of siRNAs as tools in gene silencing. Chemically synthesized siRNAs, including traditional 19-mers with 2-nt 3' overhangs, longer duplexes with blunt or 3' overhangs, and asymmetric duplexes with a blunt end and a 2-nt 3' overhang, can evoke strong dose-dependent interferon-alpha (IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) release in human peripheral blood mononuclear cells (PBMCs). This response is independent of retinoic acid-inducible gene I but may involve endosomal toll-like receptors (TLRs). The immunostimulatory effect of the siRNAs is directly related to either or both of the strands of the duplex in a sequence-dependent manner. However, although some single-stranded RNAs and siRNAs potently evoked both IFN-alpha and TNF-alpha induction, these responses were not always coupled. In accordance with this, specific chemical modifications differentially altered cytokine production, suggesting recruitment of different TLRs in a sequence-dependent manner.     

In vitro production of IgE by human peripheral blood mononuclear cells. III. Demonstration of a circulating IgE-bearing cell involved in the spontaneous IgE biosynthesis.

The presence of surface membrane IgE (SmIgE)-bearing cells in the peripheral blood (PB) of atopic patients was investigated by the use of isotype-specific rosettes of human red blood cells coupled to immunosorbent-purified rabbit or monoclonal mouse antibodies against human IgE (R or M anti-epsilon-HRBC). After dissociation of cell bound IgE by treatment with acid buffer, 2.1 +/- 0.3% and 1.2 +/- 0.3% circulating non-T, non-phagocytic, cells from atopic patients were still capable of forming rosettes with R or M anti-epsilon-HRBC, respectively. IgE molecules detectable on cells after dissociation of cytophilic IgE were quite resistant, like surface membrane IgM (SmIgM), to treatment with proteolytic enzymes, but they were removed under capping conditions by soluble anti-IgE antisera. All SmIgE-bearing (IgE+) cells also bore DR determinants, but many of them lacked SmIgM. Depletion of IgE+ cells strongly reduced the ability of PB lymphocyte suspensions from atopic patients to produce spontaneously IgE protein in vitro. Likewise, depletion of cells bearing DR determinants (DR+ cells) resulted in a marked decrease of the spontaneous IgE biosynthesis, whereas depletion of SmIgM-bearing (IgM+) cells had no effect. These data suggest that cells mainly implicated in the spontaneous IgE production in vitro seen in atopic patients are DR+ IgE+ IgM- circulating lymphocytes.     

Temporal cytokine profiling of Francisella tularensis-infected human peripheral blood mononuclear cells

BACKGROUND AND PURPOSE: Francisella tularensis is an intracellular bacterium known to replicate in monocytes and macrophages and cause tularemia in humans. Because of its infectious nature, F. tularensis is considered a biowarfare agent. Early cytokine profiles of Francisella-infected human peripheral blood mononuclear cells were evaluated. METHODS: Populations of human peripheral blood mononuclear cells were infected in vitro with F. tularensis live vaccine strain at a very low multiplicity of infection of 1:10 (bacteria:cells). A multiplex bead kit which analyzes 30 cytokines, chemokines and growth factors was utilized to measure secreted cytokines in cell supernatants 1, 4, 8, 16, and 24 h post-infection. RESULTS: Compared with uninfected controls, infected cells showed no increase in cytokine secretion at 1 and 4 h, implying a threshold for activation of immune responses. Starting at 8 h post-infection and continuing through to 24 h, an array of cytokines and growth factors was secreted by the infected cells. Some cytokines not previously associated with Francisella infection in humans were detected at 8 h, including interleukin-17 and interleukin-1 receptor agonist and vascular endothelial growth factor. CONCLUSIONS: The cytokine profiles of F. tularensis-infected peripheral blood mononuclear cells indicate an intricate pattern of both pro- and anti-inflammatory responses, including early T-cell activation.     

人外周血单个核细胞向肝样细胞转化的体外诱导培养方法*

背景：外周血单个核细胞在体外条件下可向肝样细胞转化。 目的：探索体外诱导外周血单个核细胞向肝样细胞分化的培养方法。 方法：采用密度梯度离心法联合贴壁法纯化肝硬化患者外周血单个核细胞,以含巨噬细胞集落刺激因子、白细胞介素3及β-巯基乙醇培养基培养6 d后,诱导组用含肝细胞生长因子,成纤维细胞生长因子4的培养基培养14 d;以未诱导培养的单个核细胞及L02肝脏细胞系为对照。 结果与结论：外周血单个核细胞呈透明圆形、类圆形,用巨噬细胞集落刺激因子、白细胞介素3及 β-巯基乙醇培养基培养6 d后,部分细胞向成纤维样细胞生长,诱导后部分细胞呈多边形,多角形,14 d后细胞形态逐渐接近肝细胞,并表达白蛋白、甲胎蛋白、角蛋白18。说明在一定的诱导条件下,外周血单个核细胞在体外可以向肝样细胞分化。 关键词：肝样细胞;外周血;单个核细胞;诱导;分化;干细胞 doi:10.3969/j.issn.1673-8225.2012.10.018     

In vitro and ex vivo effect of tiaprofenic acid on human peripheral blood mononuclear cells.

The effect of the non-steroidal anti-inflammatory drug (NSAID) tiaprofenic acid on different human immune parameters was investigated in vitro or following in vivo administration in healthy adult volunteers. Results from the in vitro study demonstrated an increased mitogen-induced blastogenesis and interleukin 2 (IL-2) production together with a reduced polyclonal immunoglobulin (Ig) secretion in the presence of the drug. Results from the ex vivo study showed that treatment with tiaprofenic acid had no significant effects on the immune parameters investigated, i.e. unstimulated and mitogen-induced proliferation and IL-2 production, spontaneous and stimulated Ig synthesis, lymphocyte subpopulations, serum Ig and complement levels.