Human peripheral blood mononuclear cells: A review of recent proteomic applications

Human peripheral blood mononuclear cells (PBMCs) represent a sentinel blood sample which reacts to different pathophysiological stimuli in the form of immunological responses/immunophenotypic changes. The study of molecular content of PBMCs can provide better understanding of immune processes giving the possibility of monitoring the health conditions of the host organism. Proteomic analysis of PBMCs can achieve mentioned goal as important immune-related biomarkers are easily accessible for analysis. PBMCs have been gaining attention in different research areas including preclinical or clinical investigations. In this review, recent applications of proteomic analysis of PBMCs are described and discussed. Approaches are divided based on different proteomic workflows such as in-gel, in-solution and on-filter modes. The effect of various diseases such as autoimmune, cancer, neurodegenerative, viral, metabolic, and various immune stimulations such as radiation, vaccine, corticosteroids over PBMCs proteome, are described with emphasis on promising protein biomarker candidates.     

Proteomic map of peripheral blood mononuclear cells

In the field of proteomics extensive efforts have been focused on the knowledge of proteins expressed by different cell types. In particular, enormous progress has been done in the characterization of blood cellular components. In this work, we have established a public 2-DE database for human peripheral blood mononuclear cells (PBMCs) proteins. Two hundred and forty-six spots corresponding to 174 different proteins have been identified on 2-DE gels from PBMCs isolated from six healthy individuals. All the identified proteins have been classified in thirteen categories on the basis of their differential functions or cellular localization and annotated at the http://physiology.unile.it/proteomics. The role of several proteins has been discussed in relation to their biological function. We intend to show the potentiality of PBMCs to investigate the proteomics changes possibly associated with a large number of pathologies such as autoimmune, neurodegenerative and cancer diseases.     

Effect of conjugated linoleic acid on proliferation and cytokine expression of bovine peripheral blood mononuclear cells and splenocytes ex vivo

Twenty-five primiparous Holstein cows were divided into five experimental groups (five animals per group) by different feeding (control fat preparation [CON] or conjugated linoleic acid [CLA] supplement) and slaughtering times. The daily consumption of CLA was 6.0 g of the trans-10, cis-12 CLA-isomer and 5.7 g cis-9, trans-11 CLA isomer. An initial group (IG) was slaughtered one day post partum (pp) and the remaining 20 animals after 42 and 105 days pp, respectively. Blood for peripheral blood mononuclear cells (PBMC) separation was taken seven days ante partum and immediately before slaughter. The spleen was removed during dissection for isolation of splenocytes and samples for histopathological examination. Cell viability and Concanavalin A-stimulated proliferation was analysed by MTT and Alamar Blue assay. Basal expression of cytokines (interleukin [IL]-4, IL-10, IL-12, tumour necrosis factor α [TNF-α] and interferon γ [IFN-γ]) was measured by quantitative real time polymerase chain reaction (qRT-PCR) in unstimulated PMBC and splenocytes. With PBMC, stimulation indices increased from 1 day pp to 105 days pp with no differences between CLA and CON groups. With splenocytes, the stimulation index of the CLA group was lower compared to CON group 105 days pp. Baseline expression of cytokines was not effected by CLA feeding comparing similar time points. Also, no differences occurred in the expression of IL-4 in PBMC and IL-10 as well as TNF-α in both cell populations, when comparing the feeding groups separately with IG. IL-4 was more frequently expressed in CLA group 42 days pp in splenocytes. IFN-γ expression was increased 105 days pp in CLA group in splenocytes and PBMC. IL-12 was higher expressed 105 days (PBMC) or 42 days pp (splenocytes) when compared to IG. There was no effect of CLA feeding or slaughter time on histopathology of the spleen. In conclusion, the present results demonstrate an inhibiting effect of CLA on the mitogen-induced activation of splenocytes.     

Gene expression profiles in human peripheral blood mononuclear cells as biomarkers for nutritional in vitro and in vivo investigations

Identification of chemopreventive substances may be achieved by measuring biological endpoints in human cells in vitro. Since generally only tumour cells are available for such investigations, our aim was to test the applicability of peripheral blood mononuclear cells (PBMC) as an in vitro primary cell model since they mimic the human in vivo situation and are relatively easily available. Cell culture conditions were refined, and the basal variation of gene expression related to drug metabolism and stress response was determined. Results were compared with profiles of an established human colon cell line (HT29) as standard. For biomarker development of nutritional effects, PBMC and HT29 cells were treated with potentially chemopreventive substances (chrysin and butyrate), and gene expression was determined. Key results were that relevant stress response genes, such as glutathione S-transferase T2 (GSTT2) and GSTM2, were modulated by butyrate in PBMC as in HT29 cells, but the blood cells were less sensitive and responded with high individual differences. We conclude that these cells may serve as a surrogate tissue in dietary investigations and the identified differentially expressed genes have the potential to become marker genes for population studies on biological effects.     

Human mature endothelial cells modulate peripheral blood mononuclear cell differentiation toward an endothelial phenotype

Circulating endothelial progenitor cells (EPCs) can contribute to neovascularization, even if the mechanisms by which they interact with mature endothelial cells remain unclear. The interactions between human coronary artery endothelial cells (HCAECs) and peripheral blood mononuclear cells (PBMCs) during their early differentiation towards an EPC phenotype were investigated. A co-culture model, in which the two cell types share the same culture medium in the absence of any exogenous angiogenic stimulus, was used. The role of hypoxia was assessed by pretreating HCAECs with 3% O₂ before co-culture setting. Since we have previously shown that both adherent and suspended PBMCs display a significant increase in endothelial marker expression within the 2nd day of culture in an angiogenic environment, the role of HCAECs on early PBMC differentiation was evaluated in both adherent and suspended cell fractions.A 3-day co-culture period increased the expression of VEGF-R2, VE-cadherin, α_vβ₃- and α₅-integrin in both the adherent and suspended PBMCs, assessed by cytofluorimetric analysis, and up-regulated VEGF-R1 mRNA assessed by real-time RT-PCR. HCAECs influenced PBMC adhesion, transendothelial migration and cell organization on Matrigel. Hypoxia modulated either PBMC differentiation or their functional properties. These data strongly suggest that endothelium may support the differentiation of PBMCs into EPCs.     

Recovery and functionality of cryopreserved peripheral blood mononuclear cells using five different xeno-free cryoprotective solutions

In this study, we compared three commercially available and two widely used CPAs for their ability of cryopreserving PBMCs. Similar survival (81.0%) and recovery rate (73.7%) were observed among cells using these five CPAs. However, all the cryopreserved PBMCs exhibited a significantly lower survival rate when compared with the fresh samples (94.3%). We further evaluated effector cell subpopulation and tumoricidal activity of PBMC-derived cytokine-induced killing (CIK) cells and natural killing (NK) cells. Similar and high survival (CIK: 88.6%; NK: 87.5%) and recovery (CIK: 99.5%; NK: 99.7%) rates were detected in CIK and NK cells prepared from cryopreserved PBMCs using the five CPAs. The CD3⁺CD56⁺ effector percentage (27.3%) of cryopreserved PBMC-derived CIK cells using the five different CPAs and their tumoricidal activities on melanoma CHL-1 cells (45.7%) and bladder cancer cell line T-24 (44.7%) were similar but significantly lower than those of the fresh PBMC-derived controls (effector: 30.7%; CHL-1: 84.2%; T-24: 82.2%). Cryopreserved PBMC-derived NK cells also exhibited similar tumoricidal activities (CHL-1: 73.8%; T-24: 71.9%) but was significantly lower than that of the fresh control group. We were not able to identify a specific CPA that performed superior than others in PBMC cryopreservation.     

Effect of extremely-low-frequency pulsed magnetic fields on the mitogenic response of peripheral blood mononuclear cells

The effect of extremely-low-frequency pulsed magnetic fields (PMF) on the response of human peripheral blood mononuclear cells to mitogenic stimulation is reported. We investigated 25 healthy control subjects. Mitogen-stimulated mononuclear cells were exposed to PMF for 72 h and an inhibition of 3H-thymidine uptake was observed in all but one subject. The degree of inhibition of 3H-thymidine uptake was as much as 60%. There was no significant difference between the blastogenic responses of mononuclear cells exposed to PMF for 12 h and control cultures. This study establishes an inhibitory effect of PMF on an in vitro measure of immune function.     

Impacts of CLA and dietary concentrate proportion on blood metabolite concentration and proliferation of peripheral blood mononuclear cells of periparturient dairy cows

The study aimed to examine effects of supplemented CLA to periparturient dairy cows receiving different concentrate proportions antepartum (a.p.) to investigate CLA effects on metabolism and immune function. Compared with adapted feeding, high-concentrate diet a.p. should induce a ketogenic metabolic situation postpartum (p.p.) to better understand how CLA works. A total of 64 pregnant German Holstein cows had ad libitum access to partial mixed rations based on concentrate and roughage 3 weeks before calving until day 60 p.p. A.p., cows received 100 g/day control fat (CON) or a CLA supplement, either in a low-concentrate (20%, CON-20, CLA-20) or high-concentrate diet (60%, CON-60, CLA-60). P.p., concentrate proportion was adjusted to 50% while fat supplementation continued. After day 32 p.p., half of the animals of CLA-groups changed to CON supplementation (CLA-20-CON, CLA-60-CON). A ketogenic metabolic state p.p. was not achieved and respective impacts of CLA could not be examined. Blood samples for isolation of peripheral blood mononuclear cells (PBMC) were collected on day −21, 7, 28 and 56 relative to calving. Blood chemistry samples were taken over the entire experimental period. Mitogen-stimulated proliferation of PBMC remained unaffected. Besides serum concentrations of triglycerides, total bilirubin, total protein, albumin and IGF-1, clinical-chemical serum characteristics remained uninfluenced by treatments. No post-supplementation effect could be observed. Measured blood metabolites and mitogen-stimulated proliferation of PBMC indicate that all groups had an increased metabolic stress around calving, whereby group CLA-20 was affected more severely. Overall, supplemented CLA did not positively affect metabolism or immune function of periparturient dairy cows. However, feeding CLA in a low-concentrate diet a.p. seems to increase liver stress around calving via reduced DMI.     

不同诱导因子对人外周血单个核细胞P2X7受体表达的作用

ATP激活P2X7受体可产生一系列的白细胞功能反应,因此P2X7受体的表达调控引起我们的兴趣。然而P2X7受体在正常人外周血单个核细胞(peripheral blood mononuclear cells,PBMC)、单核细胞中的表达调控机制尚未阐明。本文用半定量RT-PCR方法检测多种细胞因子、细菌抗原、丝裂原对P2X7受体表达的诱导作用,探索P2X7受体的诱导表达模式。结果表明,单个核细胞和单核细胞可检出P2X7受体的表达;白细胞介素2、4、6(interleukin-2、-4、-6,IL-2、IL-4、IL-6)、肿瘤坏死因子仪(tumour necrosis factor-α,TNF-α)等细胞因子和金黄色葡萄球菌CowanⅠ株(Staphylococcus aureus Cowan strainⅠ,SAC)、脂多糖(lipopolysaccharide,LPS)能上调PBMC的P2X7受体表达,而γ干扰素(interferon-γ,IFN-γ)、粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)、巨噬细胞集落刺激因子(macmphage colony-stimulating factor,M-CSF)和植物血凝素(phytohemagglutinin-M,PHA-M)等则没有作用;LPS和M-CSF可以提高单核细胞的P2X7受体表达,IFN-γ、TNF-α、GM-CSF作用较弱,但是这些因子的预处理并不能增强LPS对P2X7受体表达的诱导。炎症因子促进P2X7受体的表达,提示P2X7受体可能在对抗细菌感染的免疫反应中起一定作用,这有待于进一步研究。     

Adult peripheral blood mononuclear cells transdifferentiate in vitro and integrate into the retina in vivo

Adult peripheral blood-derived cells are able to differentiate into a variety of cell types, including nerve cells, liver-like cells and epithelial cells. However, their differentiation into retina-like cells is controversial. In the present study, transdifferentiation potential of human adult peripheral blood mononuclear cells into retina-like cells and integration into the retina of mice were investigated. Freshly isolated adult peripheral blood mononuclear cells were divided into two groups: cells in group I were cultured in neural stem cell medium, and cells in group II were exposed to conditioned medium from rat retinal tissue culture. After 5 days, several distinct cell morphologies were observed, including standard mononuclear, neurons with one or two axons and elongated glial-like cells. Immunohistochemical analysis of neural stem cell, neuron and retina cell markers demonstrated that cells in both groups were nestin-, MAP2 (microtubule-associated protein)- and GFAP (glial fibrillary acidic protein)-positive. Flow cytometry results suggested a significant increase in nestin-, MAP2- and CD16-positive cells in group I and nestin-, GFAP-, MAP2-, vimentin- and rhodopsin-positive cells in group II. To determine survival, migration and integration in vivo, cell suspensions (containing group I or group II cells) were injected into the vitreous or the peritoneum. Tissue specimens were obtained and immunostained 4 weeks after transplantation. We found that cells delivered by intravitreal injection integrated into the retina. Labelled cells were not detected in the retina of mice receiving differentiated cells by intraperitoneal injection, but cells (groups I and II) were detected in the liver and spleen. Our findings revealed that human adult peripheral blood mononuclear cells could be induced to transdifferentiate into neural precursor cells and retinal progenitor cells in vitro, and the differentiated peripheral blood mononuclear cells can migrate and integrate into the retina in vivo.     

Development and validation of a quantitative method for thymidine phosphorylase activity in peripheral blood mononuclear cells

Abstract

The enzyme thymidine phosphorylase (TP) is important for activation of capecitabine and 5-fluorouracil. Assessment of TP phenotype might be suitable for identification of patients at risk of fluoropyrimidine-induced toxicity. In this paper, we describe the development and validation an assay for TP activity in peripheral blood mononuclear cells (PBMCs). The assay was based on ex vivo conversion of the TP substrate thymidine to thymine. The amount of thymine formed was determined by high-performance liquid chromatography – ultraviolet detection (HPLC-UV) with 5-bromouracil as internal standard. Lymphocytes and monocytes were purified from isolated PBMCs to examine cell-specific TP activity. TP activity in PBMCs demonstrated Michaelis-Menten kinetics. The lower limit of quantification was 2.3?µg PBMC protein and assay linearity was demonstrated up to 22.7?µg PBMC protein. Within-day and between-day precisions were ≤9.2% and ≤6.0%, respectively. Adequate stability TP activity was demonstrated after long-term storage of PBMC dry pellets and lysates at ?80?°C. In monocytes, TP activity was approximately 3 times higher than in lymphocytes. Clinical applicability was demonstrated in samples that were collected from five cancer patients. A simple, precise and sensitive HPLC-UV assay for quantification of TP activity in PBMCs was developed that can be applied for clinical research.     

Immunological activity of lipopolysaccharide of Helicobacter pylori on human peripheral mononuclear blood cells in comparison to lipopolysaccharides of other intestinal bacteria

Abstract Lipopolysaccharide of Helicobacter pylori was tested for its mitogenicity and for its ability to stimulate cytokine release in human peripheral blood mononuclear cells (PBMC) of healthy and H. pylori -infected blood donors. Mitogenicity in PBMC induced by H. pylori LPS was similar to that induced by Campylobacter jejuni lipopolysaccharide, but lower than that induced by Escherichia coli lipopolysaccharide in the H. pylori negative blood donor group. Furthermore, H. pylori LPS was able to induce tumour necrosis factor (TNF) interleukin 1 (IL-1) and interleukin 6 (IL-6) secretion of PBMC. Compared with the ability of C. jejuni and E. coli lipopolysaccharides to stimulate cytokine release, H. pylori lipopolysaccharide induced a significantly lower TNF and IL-1 secretion of PBMC than the other tested bacterial lipopolysaccharides. Similar amounts of IL-6 release were obtained by stimulation of PBMC with H. pylori and C. jejuni lipopolysaccharides, whereas a higher IL-6 release was measured by stimulation with E. coli lipopolysaccharide. The results of this study suggest that H. pylori lipopolysaccharide has a lower immunological activity than lipopolysaccharides of other intestinal bacteria. This is probably due to its unusual acylation and phosphorylation pattern of lipid A.     

Apoptosis of human peripheral blood mononuclear cells by wild‐type measles virus infection is induced by interaction of hemagglutinin protein and cellular receptor,SLAM via caspase‐dependent pathway

Although MV infection causes lymphopenia and degradation of cell‐mediated immunity, the mechanisms are poorly known. MV interacts with cellular receptors which mediate virus binding and uptake and are on the surface of PBMC. In this study, apoptosis of MV‐infected PBMC in vitro was analyzed. Both PBMC treated with UV‐inactivated viruses and those infected with live MV underwent apoptosis. Apoptosis of wild‐type MV‐infected PBMC was blocked by anti‐SLAM and anti‐MV hemagglutinin antibodies, respectively. Furthermore, addition of soluble MV hemagglutinin recombinant protein induced apoptosis in PBMC. These data suggest that induction of apoptosis in MV‐infected PBMC is triggered by interaction between hemagglutinin protein of MV and receptor, without other viral components. To further determine the mechanisms of apoptosis, caspase activity was analyzed by Western blotting. Wild‐type virus Yonekawa strain‐induced apoptosis was blocked by pretreatment with pan‐caspase inhibitor (Z‐VAD‐fmk). Intriguingly, the laboratory‐adapted Nagahata strain‐induced apoptosis was not blocked by Z‐VAD‐fmk, indicating that there may be different apoptosis pathways which depend on the viral receptors, SLAM and CD46. Both extrinsic and intrinsic apoptotic pathways, including activation of caspase‐3, ‐8 and ‐9, are involved in Yonekawa strain‐induced apoptosis. Taken together, the findings of this study could open up a new avenue for understanding the molecular mechanisms of MV‐induced PBMC apoptosis and immunosuppression.     

Evaluation of influenza vaccine‐induced cell‐mediated immunity: Comparison between methods using peripheral blood mononuclear cells and whole blood

Assessment of cell‐mediated immunity (CMI) may be critical to evaluating the ability of individuals to protect themselves against influenza virus infection. However, it has been difficult to evaluate CMI because no simple means of measuring it is currently available. The aim of this study was to compare the performance of a CMI measurement method developed by us, which involves reacting whole blood with antigen, with the conventional method, which is based on isolating peripheral blood mononuclear cells (PBMCs). Correlations between these methods before and after vaccination of 26 healthy adults (aged 28–58 years; 12 men and 14 women) were assessed and changes in CMI after influenza vaccination in PBMCs cultured with antigen for 48 and 96 hr and whole blood cultured with antigen for 48 hr were studied. Results of CMI measurement using whole blood on Day 2 and PBMCs on Day 4 were found to be correlated. Spearman's correlation coefficients with four antigens (A [H1N1], A [H3N2], B [Yamagata lineage], and B [Victoria lineage]) before vaccination were 0.55, 0.61, 0.58, and 0.70, respectively and 0.40, 0.45, 0.62, and 0.52, respectively, after vaccination. CMI was detected sooner when whole blood was reacted with antigen than when PBMCs were reacted with antigen. The rate of positive reaction of influenza A (H1N1 and H3N2) in whole blood on Day 2 was higher than that in PBMCs on Day 2. Our method is simple and may be useful for vaccine development because it can measure CMI in a small amount of blood without separating off PBMCs.     

Effect of cocaine and morphine on neutral endopeptidase activity of human peripheral blood mononuclear cells cultured with lectins

We have tested the effect of alkaloids (cocaine, morphine) and enkephalins on neutral endopeptidase of peripheral blood mononuclear cells activated by lectins. When treated with concanavalin A and cocaine, peripheral blood mononuclear cells showed an enhanced activity (+110 per cent) of the membrane neutral endopeptidase, which was not related to the expression of the common acute lymphoblastic leukemia antigen at the cell surface, although both molecules have the identical amino acid sequence. Phytohemagglutinin-P, morphine and synthetic enkephalins did not induce the activity of neutral endopeptidase nor the expression of common acute lymphoblastic leukemia antigen. Our findings suggested that the drugs of abuse, cocaine and morphine, affected specific membrane constituents without altering proliferation, subcellular localization of membrane enzymes or the surface immune phenotype of peripheral blood mononuclear cells.     

Increased urinary excretion of zinc and copper by mercuric chloride injection in rats

The effects of HgCl₂ on urinary excretion of Zn, Cu and metallothionein at different time intervals were observed in male Wistar rats. The rats were given a daily intraperitoneal injection of²⁰³HgCl₂ (0.5 or 1.0 mg Hg kg^–1) for 2 days.²⁰³Hg, Zn, Cu and metallothionein in urine, kidney and liver were analyzed. Significant increases in urinary Zn and Cu concentrations were found in HgCl₂-dosed groups. Elevated urinary Zn and Cu concentrations were accompanied by an increased metallothionein excretion in urine at different time periods. Zn concentration in urine remained elevated during the entire observation period of 7 days. There were also increased concentrations of Cu and Zn in the renal cortex in one of the two exposed groups. The results indicate that urinary Cu and Zn are related to the manifestation of renal toxicity and/or the synthesis of metallothionein in kidney induced by mercury.     

Pravastatin sodium attenuated TREM-1-mediated inflammation in human peripheral blood mononuclear cells

Pravastatin sodium on triggering receptor expressed on myeloid cell-1 (TREM-1)-mediated inflammation in human peripheral blood mononuclear cells (PBMCs) has been poorly investigated. In this study, we isolated PBMCs from the peripheral blood samples of patients with chronic obstructive pulmonary disease, treated the cells with pravastatin sodium, and determined a concentration at which more than 90% cells could survive. Then we treated cells with 10?ng/ml of lipopolysaccharide, added with 10, 50, 100?μM of pravastatin sodium combined with or without LR-12, a known TREM-1 inhibitor. The expression of TREM-1 was determined by quantitative RT-PCR. The levels of TREM-1, IL-6, and TNF-α in cell culture supernatant were measured with ELISA. Simultaneously, NF-κB signaling pathway-related protein p-p65 and p-IκBα were detected by Western blot assay. Results demonstrated that pravastatin sodium significantly mitigated lipopolysaccharide-stimulated TREM-1 over-expression at mRNA and protein levels dose-dependently. Elevated IL-6 and TNF-α levels changed synchronously. LR-12 inhibited the TREM-1 over-expression and inflammatory factor production but did not show extra synergistic effect to pravastatin. Lipopolysaccharide induced phospho-p65 and -IκBα over-expression was weakened significantly when cells were treated with pravastatin sodium. In conclusion, pravastatin could inhibit TREM-1-medieted inflammation and NF-κB signaling pathway was involved.