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 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.     

Chemokine production by peripheral blood mononuclear cells in elderly subjects

The function of chemokines in promoting and modulating leukocyte migration is essential for a prompt and efficacious inflammatory response and in host defence against infections. In order to investigate whether this important aspect of immunological response is influenced by ageing, we evaluated the basal levels as well as the ability of peripheral blood mononuclear cells from young and healthy elderly subjects to produce chemokines (IL-8, MCP-1, MIP-Iα, RANTES) in response to stimulation with anti-CD3 monoclonal antibody and lipopolysaccharide (LPS), a gram negative bacterial endotoxin. Our main findings are a spontaneous chemokine production; a 20% decrease of proliferative response to anti-CD3 monoclonal antibody accompanied by an age related increase of MIP-Iα and RANTES production and by a general increase of all chemokine production compared to unstimulated conditions; a proliferative defect of monocytes to LPS challenge associated with an increase of chemokine production compared to basal conditions with a progressive age-related increase of MIP-lα. In conclusion, this study suggests that chemokines could have a compensatory role in balancing the impaired mechanisms involved in ‘specific’ immune response during ageing. The successful activation of this strategy could contribute to the good performance of immune system so maintaining healthy status in elderly.     

Chemokine production by peripheral blood mononuclear cells in elderly subjects

The function of chemokines in promoting and modulating leukocyte migration is essential for a prompt and efficacious inflammatory response and in host defence against infections. In order to investigate whether this important aspect of immunological response is influenced by ageing, we evaluated the basal levels as well as the ability of peripheral blood mononuclear cells from young and healthy elderly subjects to produce chemokines (IL-8, MCP-1, MIP-Ialpha, RANTES) in response to stimulation with anti-CD3 monoclonal antibody and lipopolysaccharide (LPS), a gram negative bacterial endotoxin. Our main findings are a spontaneous chemokine production; a 20% decrease of proliferative response to anti-CD3 monoclonal antibody accompanied by an age related increase of MIP-Ialpha and RANTES production and by a general increase of all chemokine production compared to unstimulated conditions; a proliferative defect of monocytes to LPS challenge associated with an increase of chemokine production compared to basal conditions with a progressive age-related increase of MIP-lalpha. In conclusion, this study suggests that chemokines could have a compensatory role in balancing the impaired mechanisms involved in 'specific' immune response during ageing. The successful activation of this strategy could contribute to the good performance of immune system so maintaining healthy status in elderly.     

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.     

Cytokine production by peripheral blood mononuclear cells following birch-pollen immunotherapy

We studied the Th2/Th1 balance by short-term stimulation of peripheral blood mononuclear cells (PBMC) isolated during the pollen season from seven allergic patients treated with conventional birch-pollen immunotherapy (IT) for 18 months, eight matched allergic control patients and 10 non-atopic individuals. The PBMC were cultured for 7 days with birch-pollen extract (BPE) or tetanus toxoid (TT), and then restimulated with PHA and PMA to induce high IL-5, IL-10 and IFN-gamma production. The serum levels of birch-pollen-specific IgG and IgG4 were significantly elevated after IT treatment. The proliferative response to BPE was significantly enhanced in the allergic control group, but not in the IT-treated group, compared to the non-atopic group (P<0.05). Birch-pollen-specific IL-5 production was significantly enhanced in both the IT-treated group and the allergic control group (P<0.01-0. 05). Furthermore, both the IT-treated group and the allergic control group had a cytokine profile to BPE significantly more Th2 polarized (high IL-5/IFN-gamma ratio) than to TT (P<0.05 and P<0.01, respectively). No differences in IL-10 production between the three study groups were observed. The Th2/Th1 balance in vitro correlated with the serum concentrations of birch-pollen-specific IgE (r=0.60, P<0.05), and in the IT-treated group, also with the IgG and IgG4 levels (r=0.79, P<0.05 and r=0.86, P<0.05, respectively). We conclude that conventional birch-pollen IT does not lead to changes in the cytokine profile of the circulating pool of allergen-specific T cells during birch-pollen season. However, induction of peripheral T-cell tolerance and increased production of specific IgG and IgG4 might be part of the mechanisms of IT.     

IgE production in vitro by peripheral blood mononuclear cells of patients with parasitic helminth infections.

Helminth parasites induce production of high levels of IgE antibodies but the immunoregulatory mechanisms determining this IgE biosynthesis are poorly understood. To investigate these mechanisms, peripheral blood mononuclear cells were obtained from six normal controls, six atopic patients and eight patients with parasitic helminth infections (three with schistosomiasis, two with loiasis, three with onchocerciasis). Cells were cultured at 1 X 10(6) cells/ml for 8 days in the presence of media alone or media supplemented with pokeweed mitogen (PWM) or cycloheximide; the supernatant fluids from these cultures were then assayed quantitatively for total and parasite specific IgE and IgG using an avidin-biotin amplified (for IgE) or standard (for IgG) microelisa assay. The geometric mean spontaneous IgE production was markedly elevated in peripheral blood mononuclear cells from parasitized individuals (2,487 pg/ml) when compared to those from atopics (358 pg/ml) or normals (152 pg/ml). Spontaneous IgG synthesis was equivalent in all three groups (range 140-420 ng/ml). PWM did not induce IgE production in any group and in the parasitized group even caused significant suppression of total IgE synthesis. Antigen specific antibody production (both IgE and IgG) paralleled total immunoglobulin synthesis. These findings demonstrate for the first time spontaneously enhanced IgE production in vitro in patients with helminth infections and provide a model system for studying the suppressive and regulatory mechanisms controlling IgE secretion.     

OK-432-induced cytokines production by peripheral blood mononuclear cells]

Cytokines production by OK-432-stimulated peripheral blood mononuclear cells (PBMC) were measured to investigate the in vitro function of macrophages (M phi) and lymphocytes. PBMC (1 x 10(6) cells/ml) were cultured with OK-432 (0.05 KE/ml) for 72 hr at 37 degrees C under 5% CO2, then interleukin 1 beta (IL-1 beta), interleukin 2 (IL-2) and soluble IL-2 receptor (sIL-2R) levels in the culture supernatants were measured by ELISA. While there was no significant differences of IL-1 beta production between patients with chronic active hepatitis type B (CAH-B) and controls, sIL-2R production (335 +/- 219 U/ml, mean +/- SD) was significantly decreased (p < 0.001) in patients with CAH-B. On the other hand, in pregnant women, production of both IL-1 beta (6.3 +/- 3.9 ng/ml, p < 0.01) and sIL-2R (300 +/- 169 U/ml, p < 0.001) were significantly lower than those in controls (13.5 +/- 3.8 ng/ml, 969 +/- 154 U/ml). These results suggest that the expression of IL-2R alpha on lymphocytes membrane is suppressed in patients with CAH-B, and that decreased M phi function is present in pregnant women.     

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.     

IL-12 inhibits in vitro immunoglobulin production by human lupus peripheral blood mononuclear cells (PBMC)

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by polyclonal B cell activation and by the production of anti-double-stranded (ds) DNA antibodies. Given the inhibitory effects of IL-12 on humoral immune responses, we investigated whether IL-12 displayed such an activity on in vitro immunoglobulin production by SLE PBMC. Spontaneous IgG, IgG1, IgG2, IgG3 and IgM antibody production was dramatically reduced by addition of IL-12. These results were confirmed by Elispot assays detecting IgG- and anti-dsDNA-secreting cells. While IL-6 and TNF titres measured in PBMC supernatants were not modified by addition of IL-12, interferon-gamma (IFN-γ) titres were up-regulated and IL-10 production down-regulated. Since addition of IFN-γ did not down-regulate immunoglobulin production and since the inhibitory activity of IL-12 on immunoglobulin synthesis was not suppressed by anti-IFN-γ antibody, we concluded that the effect of IL-12 on immunoglobulin production was not mediated through IFN-γ. Our data also argue against the possibility that down-regulation of endogenous IL-10 production was responsible for the effect of IL-12. Thus, inhibition of IL-10 production by IFN-γ was not accompanied by inhibition of immunoglobulin production, and conversely, restoration of IL-10 production by anti-IFN-γ antibody did not suppress the inhibitory activity exerted by IL-12 on immunoglobulin production. Taken together, our data indicate that reduction of excessive immunoglobulin and anti-dsDNA antibody production by lupus PBMC can be achieved in vitro by IL-12, independently of IFN-γ and IL-10 modulation.     

Acid-labile human interferon alpha production by peripheral blood mononuclear cells stimulated by HIV-infected cells

Summary We compared the properties of interferon (IFN) induced in peripheral blood mononuclear cells (PBMC) by free infectious HIV to that induced by HIV-infected cells fixed with glutaraldehyde. While the IFN induced by HIV was a conventional IFN alpha, the IFN induced by HIV-infected cells, although sharing with IFN alpha both antigenic properties and molecular weight, was strongly inactivated by treatment at pH lower than 4. The ability to induce acid-labile IFN alpha was exerted both by the chronically—infected cell line H9/HIV and by normal PBMC infected in vitro with HIV, while infection of inducers cells with viruses other than HIV made these cells capable of inducing only acid-stable IFN alpha. The cell involved in the production of this type of IFN seems to be B-lymphocyte.Because the presence of acid-labile IFN alpha in the serum of AIDS patients has been described, we suggest that this unusual IFN derives from interaction between circulating B-lymphocytes and the HIV-infected cells.     

The effect of age of cattle on the in vitro production of interferon by peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMC) from normal cattle of different ages and from specific pathogen-free (SPF) calves, 2 to 4 weeks old, were cultured with bovine herpes virus type 1 (BHV1), parainfluenza-3 virus (PI3) and phytohaemagglutinin (PHA). The interferon (IFN) produced was characterized by acid stability and neutralizing antisera to recombinant bovine interferons. The virus preparations were presented either live or inactivated and as cell-bound virus or free virions. PBMC from cattle of all ages produced IFN-alpha when stimulated with live BHV1 and PI3 viruses. IFN-alpha was also produced with inactivated BHV1, even in cell cultures from SPF calves. However, inactivated PI3 virus failed to induce IFN in PBMC cultures from normal cattle, but approximately half of the animals, mostly calves, produced IFN-gamma spontaneously in 48 h cultures in the absence of added antigen. PHA induced IFN-gamma at an optimal concentration of 20 micrograms per ml after 3 days in culture. An age-related maturation of the IFN response was observed as PBMC from calves less than 2 weeks old produced little or no IFN when induced with either PHA or inactivated BHV1, although some IFN-alpha was produced in cultures containing live virus. Both adherent and non-adherent cells from adults and calves over 2 weeks old produced IFN on induction with inactivated BHV1 but only the non-adherent cell population produced IFN spontaneously or in response to inactivated PI3.     

In vitro regulation by muramyl dipeptide of interferon production from peripheral blood mononuclear cells of healthy volunteers

We investigated the effect of N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) on interferon (IFN)-alpha and -gamma production by peripheral blood mononuclear cells from healthy subjects. MDP, on its own, was found to lack the ability to induce IFN production. However, this synthetic adjuvant was able to modulate IFN production induced by other stimuli. In cultures from a considerable number of tested donors, MDP enhanced IFN-gamma levels induced by phytohaemagglutinin. This effect was further potentiated after depleting the PBMNC cultures of their adherent cells. In contrast, MDP significantly suppressed the Sendai virus-induced IFN-alpha and this effect was reversed following adherent cell depletion. Identical regulatory effects on IFN production were exerted by the adjuvant active analogue of MDP, namely murabutide. The adjuvant inactive stereoisomer, MDP (DD) exhibited a similar enhancing effect on IFN-gamma but had a significantly lower inhibitory activity on IFN-alpha production. The potential value of this generation of immunomodulators in the treatment of viral infections and in models for studying the regulation of IFN at the molecular level is discussed.     

Detection of adenovirus DNA in peripheral blood mononuclear cells by polymerase chain reaction assay

Adenovirus can establish persistent infections which may reactivate and cause disease in immunocompromised hosts. Lymphocytes have been postulated to serve as a site of adenoviral persistence based upon the ability to isolate adenovirus from tonsils and to detect adenovirus DNA by Southern blot hybridization in peripheral blood mononuclear cells (PBMC). To test this hypothesis, a more sensitive and specific polymerase chain reaction (PCR) assay was developed to detect adenovirus DNA. Two sets of nested primers were designed to conserved sequences in the adenovirus E1A and hexon genes. The E1A and hexon primers amplified DNA from representative adenoviral serotypes in all six adenoviral groups (A-F). Both primers detected a single copy of the adenovirus type 2 genome but were less sensitive for the group B type 35. None of 33 PBMC specimens from healthy adults and only one of 40 pediatric samples was positive (at a low level) for adenovirus DNA by nested PCR assay. In comparison, PBMC from two children with fatal adenoviral infection were both strongly positive for adenovirus DNA. It is concluded that, in contrast to a previous study, PBMC are not a common site of persistent group C adenoviral infection. In addition, assay of PBMC by the adenovirus-specific PCR may help detect early invasive disease and warrants further evaluation. J. Med. Virol. 51:182–188, 1997. © 1997 Wiley-Liss. Inc.     

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.     

Immunomodulation of Bu-Zhong-Yi-Qi-Tang on in vitro granulocyte colony-stimulating-factor and tumor necrosis factor-alpha production by peripheral blood mononuclear cells

Bu-Zhong-Yi-Qi-Tang (BZYQT) is a Chinese medicine, and has been used for the treatment of hepatocellular carcinoma (HCC) patients. At present, we still do not fully understand the effects of BZYQT on the cellular physiology. Present in vitro study demonstrated that BZYQT is capable of increasing granulocyte colony-stimulating-factor (G-CSF) and tumor necrosis factor-alpha (TNF-alpha) production by peripheral blood mononuclear cells (PBMC) in healthy volunteers and patients with HCC. The productions of G-CSF and TNF-alpha by PBMC of volunteers were significantly stimulated by more than 125 microg/ml of BZYQT. G-CSF levels stimulated by PBMC of healthy volunteers were higher than in PBMC of the HCC patients when more than 625 microg/ml of BZYQT was administrated. The reason may be due to the impaired immunologic reactivity of mononuclear cells in HCC patients. However, the production levels of TNF-alpha in HCC patients can be stimulated to levels as high as those in healthy volunteers. When adding high concentration (3.125 mg/ml) of BZYQT to the cultured PBMC, the increments of G-CSF and TNF-alpha production decreased although there were no obvious changes in the number of metabolic active PBMC changed. TNF-alpha andG-CSF are known to play important roles in the biological defensive mechanism. These findings show that BZYQT is a unique formula for the stimulation of PBMC to produce G-CSF and TNF-alpha. Administration of BZYQT may be beneficial for patients with HCC to modulate these cytokines.     

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.     

Interleukin 1 production by peripheral blood mononuclear cells from leprosy patients.

Quantitation of interleukin 1 production by adherent mononuclear cells from peripheral blood was performed in patients with tuberculoid and lepromatous forms of leprosy. Cells from patients with tuberculoid leprosy either secreted interleukin 1 spontaneously or produced amounts within the normal range in response to lipopolysaccharide stimulation. Conversely, stimulated cells from lepromatous patients failed to produce interleukin 1 in 5 of 13 (38.5%) cases.     

Transfection of human peripheral blood mononuclear cells using immunoporation

Immunoporation has been found to be able to efficiently transfect a wide range of human cultured cell lines. This report shows that peripheral blood mononuclear cells can also be efficiently transfected using immunoporation. The immunoporation of the cells with fluorescent TMR-Dextran, using Immunofect MG beads, indicates that transient holes of 5.4nm in diameter or larger are formed during immunoporation. The efficiencies of transfection of lymphocytes transfected with vectors coding for EGFP and lacZ were found to be within the range of 15-30% with high levels of cell viability of more than 90%. In addition, it was observed that mononuclear cells stimulated with PHA expressed transfected reporter genes with a higher efficiency. In conclusion, these results demonstrate that immunoporation using Immunofect MG beads can be used for the efficient transfection of primary lymphocytes with DNA or other macromolecules.