Peripheral Blood Mononuclear Cells Secretion Research Articles

Perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) modify in vitro mitogen- and antigen-induced human peripheral blood mononuclear cell (PBMC) responses

ABSTRACT Environmental contaminants perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) are present in human serum at the highest concentration among all per- and polyfluoroalkyl substances (PFAS). Serum concentrations as high as 500 ng and 3000 ng PFOA/ml have been detected in individuals living near contamination sites and those occupationally exposed, respectively. Animal and human studies indicated that PFOA and PFOS at these serum concentrations perturb the immune system. The aim of this study was to examine the effects of in vitro exposure of human peripheral blood mononuclear cells (PBMC) to 1, 10, or 100 µM PFOA or PFOS in a medium with serum (RPMI-1640 + 5% human AB serum) on the measurement of proliferation, T cell activation, generation of memory T cells, and cytokine production/secretion. In addition, these immune system parameters were assessed for PBMC in a serum-free medium (OpSFM), which was stimulated with phytohemagglutinin (PHA) (2.5 µg/ml) or influenza vaccine antigen (0.625 µg/ml Flu Ag). PFOS decreased proliferation stimulated by PHA or Flu Ag. With Flu Ag stimulation, PFOA and PFOS inhibited the generation of memory T cells in a concentration-dependent manner. In OpSFM, PFOA and PFOS produced no marked change in proliferation and no inhibition of T cell activation. Cytokines measured in the media with Luminex methodology indicated decreased PBMC secretion of IFN-γ by PFOA and PFOS in medium with serum, but no alteration in OpSFM. The results indicated that changes in immune parameters due to PFOA or PFOS following Flu Ag stimulation are medium (±serum) dependent.

Impaired toll like receptor 9 response in pulmonary tuberculosis

Background & aimInnate immune responses are important in susceptibility to pulmonary tuberculosis (TB). In order to test the hypothesis that Toll-like receptor (TLR) 2 function would be abnormal in patients with active pulmonary TB we compared the cytokine responses of peripheral blood mononuclear cells (PBMC) to innate immune ligands in a case-control study. MethodsPBMC from 19 untreated pulmonary TB patients, 17 healthy controls, and 11 treated pulmonary TB patients, were cultured for 24h with TLR 2 ligand (PAM-CSK) and other TLR ligands (muramyl dipeptide, flagellin, lipopolysaccharide (LPS), CpG oligodeoxynucleotide (CpG-ODN)). Interleukin-8 (IL-8) was estimated in the supernatant by ELISA. Messenger RNA expression for inflammatory cytokines was quantitated using real time PCR. ResultsThe important findings were (1) reduced PBMC secretion of IL-8 in response to all ligands in active TB; (2) normal to increased PBMC secretion of IL-8 in response to all ligands except CpG ODN (TLR 9 ligand) in TB patients who had recovered; (3) absence of difference in mRNA expression for a consortium of inflammatory pathway genes between healthy controls, active pulmonary tuberculosis and treated pulmonary tuberculosis patients. ConclusionThere was a generalized post-translational suppression of the IL-8 response to innate immune ligands in active TB. There appears to be a defect of TLR 9 signaling in patients with tuberculosis, the nature of which needs to be further explored.

Ovarian steroids do not affect bovine endometrial cytokine or chemokine responses to Escherichia coli or LPS in vitro.

The risk of bacterial infection of the endometrium causing uterine disease in cattle is increased in the progesterone-dominated luteal phase of the ovarian cycle, while oestrogens or oestrus are therapeutic or protective against disease. The first line of defence against bacteria, such as Escherichia coli that cause inflammation of the endometrium, is the innate immune system, which recognises bacterial lipopolysaccharide (LPS). This study tested the hypothesis that cyclic variation in ovarian hormone concentrations alters innate immune responses within the bovine endometrium. Ex vivo organ cultures of endometrium, and in vitro cultures of endometrial epithelial and stromal cells, and peripheral blood mononuclear cells (PBMCs), all mounted inflammatory responses to E. coli or LPS, with secretion of inflammatory mediators interleukin 1β (IL1β), IL6 and IL8, and increased expression of mRNA encoding IL1B, IL6, CXCL8 (IL8) and CCL5. However, these inflammatory responses, typical of innate immunity, were not affected by the stage of ovarian cycle in which the endometrium was collected for organ culture, or by exogenous oestradiol or progesterone. Although a dexamethasone-positive control reduced inflammation stimulated by E. coli or LPS, treatment with oestradiol or progesterone, or inhibitors of oestradiol or progesterone nuclear receptors, did not affect endometrial cell or PBMC secretion of IL1β, IL6 or IL8, or IL1B, IL6, CXCL8 and CCL5 gene expression. In conclusion, the stage of the oestrus cycle or ovarian steroids did not modulate the innate immune response in the bovine endometrium in vitro.

Octreotide for the treatment of systemic lupus erythematosus: Clinical effects and an in vitro study on its therapeutic mechanism

Increased serum growth hormone (GH), together with high expression of growth hormone receptor on peripheral blood mononuclear cells (PBMCs), correlates with systemic lupus erythematosus (SLE) activity, suggesting that modulation of GH signaling may affect SLE activity. We explored the effects of octreotide (OCT), an analog of somatostatin that suppresses the release of GH, in SLE. The objectives of the study were to investigate effects of OCT on the proliferative capacity and cytokine expression of PBMCs from patients with SLE and to investigate therapeutic effects of OCT in patients with SLE. PBMCs from 13 active/inactive SLE patients and 11 controls were pretreated with or without GH and cultured with OCT. The proliferation of PBMCs was assessed by MTT assay and cytokines were quantified by ELISA. We compared the clinical response of 12 patients with SLE treated with OCT (100 µg twice daily) with 12 patients treated with prednisone over three months. OCT inhibited PBMC proliferation in a dose-dependent manner and decreased the secretion of interleukin-6 (IL-6), interleukin-10 (IL-10), and interferon-gamma (IFN-γ). Patients treated with OCT demonstrated improvements in SLEDAI, dsDNA titer, complement levels, and erythrocyte sedimentation rate (ESR). OCT inhibited PBMC proliferation and PBMC secretion of IL-6, IL-10 and IFN-γ stimulated by GH. Treatment of patients with OCT resulted in clinical improvement in SLE.

Interleukin-10 secretion from CD14+ peripheral blood mononuclear cells is downregulated in patients with acne vulgaris

Acne is a common chronic inflammatory dermatosis of the pilosebaceous unit. It is characterized by seborrhoea, comedone formation and an inflammatory response consistent with defective cellular immunity to Propionibacterium acnes. The objective of this study was to investigate the immune reactivity of patients with acne compared with healthy controls by examining the response of peripheral blood mononuclear cells (PBMCs) to stimulation with P. acnes. Particular focus was placed upon measuring the production of interleukin (IL)-10, which has an established immunoregulatory role. Venous blood was collected from 47 patients with acne and 40 age- and sex-matched healthy controls with no prior history of acne. PBMCs were cultured and their cytokine response to P. acnes investigated. Proinflammatory IL-8 and tumour necrosis factor (TNF)-alpha secretion from PBMCs was higher in patients with acne when stimulated with P. acnes. In contrast, a statistically significant reduction in PBMC secretion of anti-inflammatory IL-10 in patients with acne was identified. The impaired production of IL-10 by PBMCs from patients with acne was confined to CD14+ cells presumed to be monocytes. The ability of CD14 cells from patients with acne to phagocytose P. acnes bacteria was also observed to be defective but the addition of exogenous IL-10 to PBMC cultures restored phagocytic activity. These data suggest that patients with acne have a proinflammatory cytokine milieu and crucially are unable to contain early inflammatory changes due to a specific defect in immunosurveillance, namely low monocyte IL-10 production. Our observations raise the possibility that acne therapeutics might profitably target IL-10 both as a regulator of proinflammatory cytokines and in augmenting the CD14+ cell phagocytic response.

The interleukin-17 pathway is involved in human alcoholic liver disease # †

Immune dysregulations in alcoholic liver diseases are still unclear, especially regarding alcoholic hepatitis inflammatory burst. Interleukin-17 (IL-17) is known to enhance neutrophil recruitment. We studied the IL-17 pathway in alcoholic cirrhosis and alcoholic hepatitis. Patients with alcoholic liver disease were compared with patients with chronic hepatitis C virus (HCV) infection or autoimmune liver disease and with healthy controls. IL-17 plasma levels and peripheral blood mononuclear cell secretion were assessed by enzyme-linked immunosorbent assay (ELISA) and T cell phenotype by flow cytometry. IL-17 staining and co-staining with CD3 and myeloperoxidase were performed on liver biopsy specimens. IL-17 receptor expression was studied on liver biopsies and in human hepatic stellate cells as well as their response to recombinant IL-17 by chemotaxis assays. IL-17 plasma levels were dramatically increased in alcoholic liver disease patients. Peripheral blood mononuclear cells of patients with alcoholic liver disease produced higher amounts of IL-17, and their CD4(+) T lymphocytes disclosed an IL-17-secreting phenotype. In the liver, IL-17-secreting cells contributed to inflammatory infiltrates in alcoholic cirrhosis, and alcoholic hepatitis foci disclosed many IL-17(+) cells, including T lymphocytes and neutrophils. In alcoholic liver disease, liver IL-17(+) cells infiltrates correlated to model for end-stage liver disease score, and in alcoholic hepatitis to modified discriminant function. IL-17 receptor was expressed in alcoholic liver disease by hepatic stellate cells, and these cells recruited neutrophils after IL-17 stimulation in a dose-dependent manner through IL-8 and growth related oncogen alpha (GRO-alpha) secretion in vitro. Human alcoholic liver disease is characterized by the activation of the IL-17 pathway. In alcoholic hepatitis, liver infiltration with IL-17-secreting cell infiltrates is a key feature that might contribute to liver neutrophil recruitment. (Clinical trials number NCT00610597).

Effects of melatonin on the cytokines production of neonatal cord blood mononuclear cell

Objective To investigate the effects of melatonin(MLT)on the cytokines production of neonatal cord blood mononuclear cells(CBMC)in vitro. Methods Ten blood specimens from placenta vena umbilicalis of 10 healthy term delivery newborns were obtained,and 10 healthy adult volunteers'peripheral blood mononuclear cells(PBMC)specimens were taken as controls.CBMC and PBMC suspensions were cultivated in the system without serum and hormone,and then 200 μl PBMC/CBMC suspensions(2 105 cells)were co-cultured with MLT with different concentrations(50 ng/ml,5 ng/ml,500 pg/ml,50 pg/ml)for 72 hours.The concentrations of cytokines including IL-1β,IL-2,IL-4,IL-6,IL-8,IL-12,IFN-γ and TNF-α in the cell supernatant were detected.Results The 500 pg/ml MLT induced secretions of IL-2[(12.18 7.31)pg/ml],IL-6[(2091.08±968.58)pg/ml],IL-8[(2779.04±1168.49)pg/ml],IL-12[(15.29±6.88)Pg/ml]and IFN-γ[(52.22 17.77)pg/ml]of CBMC were more than those of the control group,which were(6.99±4.94)pg/ml,(1017.06±452.60)pg/ml,(1293.33±791.34)pg/ml,(6.84±2.26)pg/ml and(22.24±10.38)pg/ml(P<0.05).Compared with control group[(175.63±62.10)Pg/ml],all concentrations of MLT could inhibit the TNF-α secretion[(24.87 11.21)pg/ml,(18.14±9.44)pg/ml,(31.38±15.28)pg/ml and(28.18±17.35)pg/ml]of CBMC significantly.The 50 ng/ml MLT could induce the PBMC's secretion of IL-2, IL-12, IFN-γ,and inhibit the secretion of TNF-α. The best effect of MLT on CBMC was at the concentration of 500 pg/ml, while that on PBMC was 5 ng/ml. Comparing the best effects of MLT, the levels of IL-12 and IFN-γ of CBMC were higher than those of PBMC, and IL-2 level of CBMC was lower than that of PBMC (P<0.01). Conclusions MLT could promote the cytokine secretion of PBMC and CBMC, and the effects of MLT on IL-12 and IFN-γ secretion of CBMC were stronger than those of PBMC. The results suggested that MLT may contribute to promote the immune function in newborn infants, and provide experimental evidence for clinical application of MLT. Key words: Melatonin; Fetal blood; Leukocytes, mononuclear; Cytokines

Dietary nucleotides and human immune cells. II. Modulation of PBMC growth and cytokine secretion

Objective The immune system is dependent on purines and pyrimidines as building blocks for DNA and RNA synthesis to enable rapid cell proliferation and protein synthesis. Emerging evidence suggests that dietary nucleotides optimize immune function. We investigated whether growth and function of human immune cells were affected by an exogenous source of nucleotides during specific antigen challenge. Methods Peripheral blood mononuclear cells from healthy individuals ( n = 10) were stimulated with influenza virus antigen and either DNA sodium from fish soft roe (DNA), RNA from bakers yeast ( Saccharomyces cerevisiae) (RNA), 2′ deoxyadenosine 5′-monophosphate sodium (dAMP), 2′ deoxycytidine 5′-monophosphate sodium (dCMP), 2′deoxyguanosine 5′-monophosphate sodium (dGMP), 2′ deoxyuridine 5′-monophosphate sodium (dUMP) or thymidine sodium (TMP). Growth effects were ascertained by measuring the amount of tritium-labeled thymidine, incorporated into cell DNA. Cell function was measured by detection of IFN-gamma, TNF-alpha and IL-10 production. Results Specific nucleotide derivatives alone did not affect the growth of healthy peripheral blood mononuclear cells. However, the nucleotide derivatives influenced immune cell growth and cytokine secretion when cocultured with specific antigen. DNA, RNA, dAMP, dCMP and dUMP increased influenza virus antigen induced immune cell proliferation. In contrast dGMP and TMP inhibited the antigen-induced growth response. RNA and dAMP cocultured with virus antigen significantly increased peripheral blood mononuclear cell secretion of IFN-gamma, IL-10 and TNF-alpha. DNA increased virus antigen-induced immune cell secretion of IFN-gamma only, whereas dUMP significantly increased secretion of IL-10 only. dGMP completely inhibited virus-triggered IFN-gamma secretion, whereas TMP did not change the virus induced secretion pattern of measured cytokines. Conclusion Nucleotide derivatives affect growth and function of specific virus antigen-stimulated human immune cells in vitro.

T-Helper 2 and 3 type immunity to trophoblast in successful in vitro fertilization-embryo transfer

To determine whether peripheral blood mononuclear cell (PBMC) secretion of T-helper (Th)-1 type cytokines and Th-2 and Th-3 type cytokines in women undergoing in vitro fertilization-embryo transfer (IVF-ET) is associated with therapeutic failure and success, respectively. Cohort study. Academic medical center. One hundred one women undergoing IVF-ET and 19 fertile controls. Peripheral blood was obtained from women undergoing IVF-ET before oocyte retrieval and from 19 nonpregnant fertile controls. The PBMCs were cultured in the presence or absence of a protein extract from either a trophoblast cell line or sperm membrane. Supernatants from PBMC cultures were tested by enzyme-linked immunoabsorbent assay (ELISA) for the Th-1 type cytokines tumor necrosis factor-alpha (TNF)-alpha) and interferon-gamma (IFN)-gamma), the Th-2 type cytokines interleukin (IL)-6 and IL-10, and the Th-3 type cytokine transforming growth factor (TGF)-beta1. Levels of IL-6 and IL-10 were significantly higher in controls than in infertile women with endometriosis, and levels of IL-10 were higher in controls than in women with unexplained infertility. No differences were found in unstimulated levels of TNF-alpha, IFN-gamma, or TGF-beta1 between infertile patients and controls. In trophoblast-stimulated PBMC cultures, levels of TGF-beta1 were significantly lower in subjects who experienced failed compared with ongoing pregnancies. Baseline PBMC secretion of IL-6 and IL-10 is higher in fertile controls than in women with endometriosis, and IL-10 secretion is also higher than in women with unexplained infertility. Trophoblast-stimulated PBMC secretion of TGF-beta1 is positively associated with the establishment of successful pregnancy in women undergoing IVF-ET. Our study provides novel evidence to support a faciliatory role of Th-2 and Th-3 type responses to trophoblast in early pregnancy.

High interleukin 12 and low interleukin 10 production after in vitro stimulation detected in sepsis survivors

To evaluate viability of isolated peripheral blood mononuclear cells (PBMC) and production of cytokines in vitro after stimulation as prognostic factors for survival in sepsis patients. Prospective study of the biological response of PBMC in the onset of severe sepsis. Research laboratory of molecular biology and immunology and university hospital ICU, Faculty of Medicine, Trakia University. Twenty-three patients meeting the criteria for severe sepsis, and 14 control subjects. Isolated PBMC were stimulated in vitro with: C3-binding glycoprotein (C3bgp; 30 microg), lipopolysaccharide (30 microg), phytohemagglutinin (20 microg), pokeweed mitogen (30 microg), and dexamethasone (500 microg). We measured the levels of interleukins (IL) 6, 10, and 12 in culture supernatants. Stimulation with C3bgp and phytohemagglutinin led to significantly lower PBMC secretion of IL-6 in nonsurvivors than in survivors and healthy donors. Stimulation with C3bgp, lipopolysaccharide, and pokeweed mitogen considerably reduced IL-12 production in nonsurvivors. Stimulation with lipopolysaccharide and pokeweed mitogen caused immune cells in nonsurvivors to produce higher levels of IL-10 than in survivors. Survival of PBMC reduced viability for nonsurvivors' PBMC, both spontaneously and as induced by lipopolysaccharide or pokeweed mitogen. The viability of PBMC at the onset of sepsis and enhanced production of IL-12 and diminished production of IL-10 after stimulation with all stimuli used may be a favorable prognostic factor in sepsis.

Marijuana components stimulate human peripheral blood mononuclear cell secretion of interferon-gamma and suppress interleukin-1 alpha in vitro

We investigated the in vitro effects of both psychoactive and nonpsychoactive marijuana components on leukocyte secretion of the immunoregulatory cytokines interleukin-1 alpha (IL-1), tumor necrosis factor alpha (TNF), interferon-gamma (IFN) and interleukin-2 (IL-2). Psychoactive delta-9-tetrahydrocannabinol (THC) and nonpsychoactive cannabidiol (CBD) were added to cultures of mitogen-activated human peripheral blood mononuclear cells (PBMC) and the concentrations of IL-1, TNF, IFN and IL-2 in culture supernatants were measured by ELISA systems. Concentrations of THC and CBD, comparable to plasma levels found after smoking marijuana (10–100 ng/ml), increased the concentration of measurable IFN (139 and 68%), while high concentrations of both cannabinoids (5–20 μg/ml) completely blocked synthesis and/or release of this cytokine. CBD was also shown to decrease the measurable quantity of both IL-1 and TNF. In contrast to the effects of IFN, IL-1 and TNF, both cannabinoids, had no effect on IL-2 secretion. This report suggests that both psychoactive and nonpsychoactive components of marijuana are immunomodulating and can potentially alter cytokine secretion of human PBMC.