Preincubation Of Peripheral Blood Mononuclear Cells Research Articles

Anti-inflammatory and antioxidant actions of extracts from Rheum rhaponticum and Rheum rhabarbarum in human blood plasma and cells in vitro

Rheum rhaponticum L. (rhapontic rhubarb) and Rheum rhabarbarum L. (garden rhubarb) are edible and medicinal rhubarb species used for many centuries in traditional medicine. This work is focused on the biological activity of extracts from petioles and roots of R. rhaponticum and R. rhabarbarum as well as rhapontigenin and rhaponticin, typical stilbenes present in these rhubarbs, in a context of their effects on blood physiology and cardiovascular health. Anti-inflammatory properties of the examined substances were evaluated in human peripheral blood mononuclear cells (PBMCs) and THP1-ASC-GFP inflammasome reporter cells. Due to the coexistence of inflammation and oxidative stress in cardiovascular diseases, the study design included also antioxidant assays. This part of the work involved the assessment of the protective efficiency of the examined substances against the peroxynitrite-triggered damage to human blood plasma components, including fibrinogen, a protein of critical importance for blood clotting and maintaining the haemostatic balance. Pre-incubation of PBMCs with the examined substances (1–50 μg/mL) considerably decreased the synthesis of prostaglandin E2 as well as the release of pro-inflammatory cytokines (IL-2 and TNF-α) and metalloproteinase-9. A reduced level of secreted apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks in the THP-1-ASC-GFP cells was also observed. The examined substances significantly diminished the extent of ONOO‾induced oxidative modifications of blood plasma proteins and lipids and normalized, or even strengthened blood plasma antioxidant capacity. Furthermore, a reduction of oxidative damage to fibrinogen, including modifications of tyrosine and tryptophan residues along with the formation of protein aggregates was found.

Endosomal Toll-Like Receptors (TLRs) mediate enhancement of IL-17A production triggered by Epstein-Barr virus (EBV) DNA in mice.

EBV has long-been associated with autoimmune disorders. We have previously demonstrated that EBV DNA increases the production of IL-17A in mice. This property may play a role in the association of EBV with autoimmune diseases. The objective of this study was to elucidate mechanisms through which EBV DNA modulates IL-17A levels in mice. To study the potential role of endosomal receptors in detecting EBV DNA, chloroquine, an endosomal maturation inhibitor, was used to treat mouse peripheral blood mononuclear cells (PBMCs) in the presence or absence of EBV DNA. IL-17A levels were then assessed by ELISA. Subsequently, to determine whether TLR3, 7 or 9 played a role in this pathway, specific inhibitors were used for these TLRs both in mouse PBMCs and in vivo in BALB/c mice treated with the viral DNA; IL-17A levels were then similarly assessed. IL-17A production was enhanced from mouse PBMCs cultured with EBV DNA; pre-incubation of PBMCs with chloroquine significantly reduced its production. When cells were cultured with EBV DNA and a TLR3, 7 or 9 inhibitor, a significant decrease in IL-17A levels was detected. A similar decrease in the EBV DNA-triggered IL-17A production in mice was observed when animals were treated with the TLR inhibitors. Endosomal TLRs appear to be involved in recognizing EBV DNA and subsequently triggering IL-17A production in mice. Targeting these receptors in EBV positive subjects with autoimmunity may be useful pending investigations assessing whether they play a similar role in humans.

Posaconazole in Vitro Action on Peripheral Blood Mononuclear Cells From Sarcoidosis Patients

SESSION TITLE: Diffuse Lung Disease 1 SESSION TYPE: Original Investigation Poster PRESENTED ON: Wednesday, November 1, 2017 at 01:30 PM - 02:30 PM PURPOSE: Treatment with anti-fungal medication is as efficient as corticosteroids in sarcoidosis in many, albeit not all, patients.Insoluble β-glucan, a component in the fungal cell wall, induced the production of several cytokines from peripheral blood mononuclear cells (PBMC) “in vitro”. Against this background two hypotheses can be formulated: anti-fungal medication influences immune cell activity and decreases the secretion of cytokines after stimulation with insoluble β-glucan and anti-fungal medication acts on insoluble β-glucan and decreases its capacity to induce cytokine production from PBMC. METHODS: Human PBMCs from sarcoidosis (n=6) and healthy subjects (n=5) were stimulated for 4h with particulate (1→3)-β-D-glucan (BGP) after 24h preincubation with posaconazole, with methylprednisolone or with medium alone. We preincubated the BGP with or without the Posaconazole for 1h before adding the PBMCs from healthy subjects for additional 4h. The viability of the cells was determined, the concentrations of TNF-α in cell culture supernatants were measured and expression of NF-kB mRNA. RESULTS: When adding the PBMCs from healthy subjects for additional 4 h to BGP preincubated either with or without Posaconazole, there was no difference in TNF-α production (884 ± 23.9 pg/ml; 981 ± 70.2 pg/ml). Meanwhile, the secretion of TNF-α was significantly higher in sarcoidosis when PBMCs were stimulated with BGP (2162 ± 432.6 pg/ml; 1338 ± 116.7 pg/ml; p=0,032) than in healthy subjects. However, in sarcoidosis patients, preincubation of PBMCs with Posaconazole significantly reduced the secretion of TNF-α after stimulation with BGP (p=0,046), while having no effect on the healthy. On the other hand, Posaconazole significantly reduced the expression of NF-Kb in both groups and there was no significant difference in cell viability in cultures. Thus, the results demonstrate that the inhibitory effect of Posaconazole on PBMCs from sarcoidosis patients is neither a cytotoxic effect of Posaconazole on PBMCs nor a result of the interaction of Posaconazole with BGP. CONCLUSIONS: We have demonstrated that posaconazole “in vitro”significantly reduces the secretion of TNF-α from sarcoidosis patients. The results are consistent with our previous clinical research.There, we have already demonstrated that antifungal treatment is as efficient as corticosteroid treatment of granulomatous inflammation in many, albeit not all,sarcoidosis patients. CLINICAL IMPLICATIONS: Additional studies are required to define the phenotype where the antifungal treatment is not efficient. DISCLOSURE: The following authors have nothing to disclose: Marjeta Tercelj-Zorman, Sanja Stopinsek, Barbara Salobir, Alojzij Ihan, Sasa Simcic No Product/Research Disclosure Information

Decreased TLR2 signal expression in peripheral blood mononuclear cell from patients with cryptococcal meningitis.

Toll-like receptors are the most important pattern recognition receptors that can recognize conserved molecular structures shared by large groups of pathogens. Here, the aim was to determine the expression and role of TLR2 in peripheral blood mononuclear cells (PBMCs) from patients with cryptococcal meningitis and healthy controls. TLR2 expression was measured using RT-PCR and western blotting. The role of TLR2 in cytokine production by PBMCs after Cryptococcus neoformans exposure was assessed in healthy controls prior to incubation with anti-TLR2. TLR2 mRNA and protein expression were both weaker in patients with cryptococcal meningitis than in healthy controls. Furthermore, pre-incubation of PBMCs from healthy donors with anti-TLR2 led to reduced expression of IFN-γ and IL-12p70, but not of IL-4 and IL-10, following C. neoformans stimulation. Our results suggest that impaired expression of TLR2 may be involved in defective host defense to C. neoformans through an attenuated Th1 response.

Bovine lactoferrin enhances proliferation of human peripheral blood lymphocytes and induces cytokine production in whole blood cultures.

Lactoferrin belongs to the immunoregulatory milk proteins involved in iron metabolism as well in providing innate immunity to newborns. The protein has been the subject of numerous clinical studies. The aim of this investigation was to evaluate the effects of bovine lactoferrins (bLF), differing in source and iron content, on spontaneous proliferation of human peripheral blood mononuclear cells (PBMC) and cytokine production by human whole blood cultures. The following bLF preparations were used: partially iron saturated or devoid of iron bLF from milk and bLF from colostrum. The study was conducted on 12 healthy volunteers (men, 20-24years old). The effects of bLFs on the proliferation of PBMC in four-day cultures was studied at 50-0.6µg/mL concentration range and the rate of proliferation was determined using the MTT colorimetric method. TNF αand IL-6 levels, induced by the bLFs in 24 hwhole blood cultures, were measured by ELISA. The lactoferrins stimulated autologous proliferation of human peripheral blood mononuclear cells (PBMC) in adose-dependent manner, with acomparable efficacy. This stimulation occurred both in the constant presence of bLFs in the cultures and also upon preincubation of PBMC with bLFs with subsequent exhaustive wash of cells. Only bLF from colostrum induced production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in cultures of whole blood cells. This phenomenon took place predominantly at concentration of 50µg/mL. The results showed potent stimulation of the proliferative response of PBMC by bovine lactoferrin, associated with the induction of proinflammatory cytokines only in the case of colostral bLF.This observation may be of importance when high doses of bLF are used in therapy and by designing diet supplementation with this protein.

Intravenous Immunoglobulins Suppress Antibody-Dependent Effector Functions of Human Peripheral Blood Cells

RationaleSeveral studies have shown the importance of self-reactive antibodies in the maintenance of autoimmune diseases. Therefore, we asked whether IGIV, which is therapeutically effective in some of those diseases, directly modulates antibody-mediated effector functions.MethodsWe used a human in vitro system to analyze potential modulatory effects of IGIV on effector functions in antibody-dependent cellular cytotoxicity (ADCC). Human peripheral blood mononuclear cells (PBMC) from 47 healthy volunteers were pre-incubated with IGIV and then added to human breast cancer cells (SK-BR3) opsonized with a specific antibody. The cytotoxic damage to SK-BR3 cells was determined by measuring lactate dehydrogenase release into supernatants. IGIV-mediated effects on viability and Fc gamma receptor (FcγR) expression of PBMC were analyzed by flow cytometry.ResultsPre-incubation of PBMC with IGIV for 48h resulted in a marked inhibition of ADCC, ranging from 15% to 74% (mean 42%) which could not be mimicked with human monoclonal IgG1 and IgG2 antibodies. Flow cytometric analysis indicated that IGIV induced natural killer (NK) cell death, which significantly correlated with ADCC inhibition. The IGIV-induced decrease in NK cell viability was not triggered by FcγR ligation since the addition of FcγR blocking antibodies could not prevent cell death induction. Furthermore, IGIV enriched for sialic acid bearing glycans by lectin affinity chromatography with Sambucus nigra agglutinin showed no differences in the effects on NK cell viability and ADCC compared with non-enriched IGIV.ConclusionsIGIV modulates antibody-dependent effector functions of human immune cells, which could contribute to the modulatory activities of IGIV in inflammatory and autoimmune diseases. RationaleSeveral studies have shown the importance of self-reactive antibodies in the maintenance of autoimmune diseases. Therefore, we asked whether IGIV, which is therapeutically effective in some of those diseases, directly modulates antibody-mediated effector functions. Several studies have shown the importance of self-reactive antibodies in the maintenance of autoimmune diseases. Therefore, we asked whether IGIV, which is therapeutically effective in some of those diseases, directly modulates antibody-mediated effector functions. MethodsWe used a human in vitro system to analyze potential modulatory effects of IGIV on effector functions in antibody-dependent cellular cytotoxicity (ADCC). Human peripheral blood mononuclear cells (PBMC) from 47 healthy volunteers were pre-incubated with IGIV and then added to human breast cancer cells (SK-BR3) opsonized with a specific antibody. The cytotoxic damage to SK-BR3 cells was determined by measuring lactate dehydrogenase release into supernatants. IGIV-mediated effects on viability and Fc gamma receptor (FcγR) expression of PBMC were analyzed by flow cytometry. We used a human in vitro system to analyze potential modulatory effects of IGIV on effector functions in antibody-dependent cellular cytotoxicity (ADCC). Human peripheral blood mononuclear cells (PBMC) from 47 healthy volunteers were pre-incubated with IGIV and then added to human breast cancer cells (SK-BR3) opsonized with a specific antibody. The cytotoxic damage to SK-BR3 cells was determined by measuring lactate dehydrogenase release into supernatants. IGIV-mediated effects on viability and Fc gamma receptor (FcγR) expression of PBMC were analyzed by flow cytometry. ResultsPre-incubation of PBMC with IGIV for 48h resulted in a marked inhibition of ADCC, ranging from 15% to 74% (mean 42%) which could not be mimicked with human monoclonal IgG1 and IgG2 antibodies. Flow cytometric analysis indicated that IGIV induced natural killer (NK) cell death, which significantly correlated with ADCC inhibition. The IGIV-induced decrease in NK cell viability was not triggered by FcγR ligation since the addition of FcγR blocking antibodies could not prevent cell death induction. Furthermore, IGIV enriched for sialic acid bearing glycans by lectin affinity chromatography with Sambucus nigra agglutinin showed no differences in the effects on NK cell viability and ADCC compared with non-enriched IGIV. Pre-incubation of PBMC with IGIV for 48h resulted in a marked inhibition of ADCC, ranging from 15% to 74% (mean 42%) which could not be mimicked with human monoclonal IgG1 and IgG2 antibodies. Flow cytometric analysis indicated that IGIV induced natural killer (NK) cell death, which significantly correlated with ADCC inhibition. The IGIV-induced decrease in NK cell viability was not triggered by FcγR ligation since the addition of FcγR blocking antibodies could not prevent cell death induction. Furthermore, IGIV enriched for sialic acid bearing glycans by lectin affinity chromatography with Sambucus nigra agglutinin showed no differences in the effects on NK cell viability and ADCC compared with non-enriched IGIV. ConclusionsIGIV modulates antibody-dependent effector functions of human immune cells, which could contribute to the modulatory activities of IGIV in inflammatory and autoimmune diseases. IGIV modulates antibody-dependent effector functions of human immune cells, which could contribute to the modulatory activities of IGIV in inflammatory and autoimmune diseases.

Abstract 1809: Stimulation of antibody dependent cell-mediated cytotoxicity by interleukin-15 is equivalent to interleukin-2

Abstract An important strategy for the treatment of cancer involves administration of tumor-specific antibodies that target tumor cells for destruction by the individual's leukocytes via antibody dependent cell-mediated cytotoxicity (ADCC). However, ADCC is frequently depressed in individuals with cancer. Interleukin-2 (IL-2), a pro-inflammatory cytokine, enhances ADCC and is used clinically for this purpose. Interleukin-15 (IL-15) is a cytokine that shares many functions with IL-2, including stimulation of ADCC and proliferation and activation of natural killer cells. In contrast to IL-2, IL-15 supports the survival of CD8+ memory T cells, does not produce T cell activation-induced cell death and has no marked effects on Treg cells. Since these different functions may prove beneficial when using a cytokine to stimulate ADCC in vivo in cancer immunotherapy, we compared the in vitro effect of IL-15 (generously provided by Dr. Stephen Creekmore, NCI-Frederick) versus IL-2 to enhance ADCC. Using a standard 51Cr release assay, M21 human melanoma cells were loaded with 51Cr and incubated for four hours at 37° with human peripheral blood mononuclear cells (PBMC), hu14.18K322A, (generously provided by Dr. Fariba Navid of St. Jude Children's Research Hospital) a monoclonal antibody specific for GD2 (disialoganglioside) that is overexpressed on M21 cells (5 ng/ml), and either media, IL-2 (10 ng/ml) or IL-15 (10 ng/ml). The ratio of effectors (PBMCs) to targets (M21) was 10:1 or 30:1 and % cytotoxicity was determined from which lytic units were calculated. Incubation with IL-2 augmented ADCC from 163±39 to 230±40 lytic units (n=6, mean±SEM) while incubation with IL-15 enhanced ADCC from 148±27 to 229±36 lytic units (n=6). ADCC response was comparable for IL-2 and IL-15 (p=0.4857) and similar results were obtained using higher and lower concentrations of hu14.18K322A (0.1 – 100 ng/ml) and IL-2 and IL-15 (5 – 25 ng/ml), and after pre-incubation of PBMCs with IL-2 or IL-15 (4 – 44 hours). These experiments indicate that IL-2 and IL-15 demonstrate comparable stimulation of ADCC in vitro. If IL-15 produces equivalent enhancement of ADCC compared to IL-2 in vivo, it may be a potential immunotherapeutic for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1809. doi:10.1158/1538-7445.AM2011-1809

Lactic acid stimulates interleukin-23 production by peripheral blood mononuclear cells exposed to bacterial lipopolysaccharide

Lactic acid is the predominant acid present in the vagina. We evaluated the consequences of lactic acid, at physiological levels present in the vagina, on cytokine responses of peripheral blood mononuclear cells (PBMCs) obtained from 10 individuals in the presence or absence of bacterial lipopolysaccharide. Preincubation of PBMCs in 15 mM lactic acid before the addition of lipopolysaccharide resulted in a 246% mean increase in interleukin-23 (IL-23) secretion over that released in the presence of lipopolysaccharide alone (P=0.0068). The lipopolysaccharide-induced production of tumor necrosis factor-α, IL-6, IL-10 and IL-12 was unaffected by lactic acid. IL-23 stimulation was not observed if the lactic acid was neutralized before its addition to the culture medium or if hydrochloric acid was substituted for lactic acid. In the absence of lipopolysaccharide, lactic acid did not stimulate the production of IL-23 or any of the other cytokines. The increase in IL-23 production was proportional to the lactic acid concentration over a 15-60 mM range. We conclude that at body sites characterized by lactic acid accumulation, such as in the human vagina, exposure to gram-negative bacteria results in selective IL-23 production, leading to a subsequent preferential stimulation of the Th17 T lymphocyte pathway.

Lenalidomide Enhances Multiple Myeloma Cytotoxicity Induced by a Novel Fc Domain-Engineered Anti-HM1.24 Monoclonal Antibody with Augmented NK Cell Degranulation

AbstractAbstract 4064HM1.24, an immunological target highly expressed on majority of multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). Recently, XmAb5592, a novel Fc-domain engineered humanized anti-HM1.24 mAb with specific Fc-domain modification, was shown to induce >10-fold antibody-dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis against MM cells, when compared with the humanized normal anti-HM1.24 IgG1 (XmAb5627) from which it is derived (ASH Abstract#609, 2009). Here we investigated whether XmAb5592, when combined with other anti-MM drugs, further enhanced ADCC against MM cell lines and primary patient MM cells using Calcein-AM release ADCC assays and flow cytometric analysis for cell membrane CD107a to specifically quantitate NK cell (CD56+CD3-) activation. Addition of lenalidomide (10 μ M) in standard ADCC assays increased XmAb5592 (0.1 μ g/ml)-induced cell lysis against MM1S, MM1R, and RPMI8226 cells in the presence of peripheral blood mononuclear cells (PBMCs) from normal donors (n=2). Specifically, XmAb5592 (0.01, and 0.1 μ g/ml) combined with lenalidomide (2.5, 5, 10 μ M), in the presence or absence of IL-2 (100 units/ml), synergistically induced NK-mediated RPMI8226 MM cell lysis, as evidenced by combination index (CI) < 1 (0.08-0.89). Pre-incubation of PBMCs with additional IL-2 (100 units/ml) enhanced even stronger XmAb5592-induced cytotoxicity against MM cells than pretreatment with lenalidomide alone. Lenalidomide also enhanced PBMC effectors to kill primary patient MM cells. Cell surface CD107a, as a functional marker for NK cell activation dependent on granzyme B secretion, was further determined following target myeloma cell incubation, with or without NK cells and in the presence of mAbs. NK cells were activated by as low as 0.001 μ g/ml of XmAb5592 only in the presence of MM1S tumor cells. In contrast, neither XmAb6166, an Fc-domain knockout of XmAb5592 without NK binding, nor XmAb4614, a similar Fc-engineered mAb targeting respiratory syncytial virus (RSV) antigen, induced any cell surface CD107a on NK cells incubated with MM cells. Specific XmAb5592-induced CD107a-dependent NK cell degranulation further correlated with IFNγ secretion (r=0.7, p=0.03). XmAb5592 induced > 10-fold more potent NK degranulation than XmAb5627, which significantly correlated with MM cell lysis by ADCC, even in the presence of bone marrow stromal cells (BMSCs). Moreover, no significant HM1.24 was expressed on NK cells stimulated with or without IFN-α, suggesting minimal NK toxicity. XmAb5592 more potently (>10-fold) than XmAb5627 induces homotypic aggregation and adhesion of NK cells, which was further enhanced by lenalidomide. Finally, minimal HM1.24 expression was confirmed on different PBMC subsets including CD15+ (PMN), CD19+ (B), CD14+ (MC), and CD3+ (T) cells. These results indicate that lenalidomide further potentiates XmAb5592-induced myeloma cell killing via NK-mediated ADCC, providing a rationale to combine both novel drugs to improve patient outcome in MM. Disclosures:Muchhal:Xencor Inc: Employment. Desjarlais:Xencor Inc: Employment. Richardson:Gentium: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

The role of TFPI in regulation of TF-induced thrombogenicity on the surface of human monocytes

Introduction Although the procoagulant reactivity of monocytes largely depends on expression and cell surface presentation of tissue factor (TF), little is known about the impact of tissue factor pathway inhibitor (TFPI) on regulation of TF function on the monocyte surface. Materials and methods Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy subjects and cryopreserved. We investigated TF and TFPI mRNA expression by reverse transcription-quantitative real-time PCR (RT-qPCR), surface presentation by flow cytometry and confocal microscopy, and TFPI-mediated regulation of TF functional activity on the surface of resting and LPS-stimulated PBMCs by TF activity assay and Calibrated Automated Thrombogram (CAT) assay. Results Unstimulated PBMCs contained nearly no TF, but detectable TFPI protein levels. TFPI mRNA levels were 2-fold higher than TF, and the TFPIα mRNA isoform expression was higher than TFPIβ. LPS stimulation caused a parallel and sustained upregulation of both TFPI isoforms, concomitant with increased surface presentation of TFPI antigen. Stronger, but transient upregulation of TF mRNA and surface antigen was observed at 6 hrs of LPS stimulation. After LPS stimulation TF and TFPI were co-localized in the same areas of the monocyte membrane. Pre-incubation of PBMCs with anti-TFPI IgG significantly enhanced TF activity, shortened Lag-time, and increased thrombin generation. TFPI-dependent inhibition of TF was more prominent in resting than in LPS-stimulated cells. Conclusions Our results support the concept that surface TFPI is an important regulator of procoagulant reactivity of human monocytes.

Binding of HCV E2 to CD81 induces RANTES secretion and internalization of CC chemokine receptor 5

Hepatitis C virus (HCV) infection has been shown to be associated with reduced expression of the CC chemokine receptor (CCR) 5, and reduced responsiveness of lymphocytes to chemokines. However, the mechanism by which HCV alters CCR5 expression remains unclear. Here, we investigated whether altered CCR5 expression in hepatitis C results from interactions of CD81 with the HCV E2 protein. Peripheral blood mononuclear cells (PBMC) from HCV-negative individuals were prepared by Ficoll density gradient separation. PBMC subpopulations (CD4+, CD8+ lymphocytes, CD19+ B cells, natural killer (NK) cells and monocyte-derived dendritic cells) were isolated and stimulated with immobilized HCV E2, and changes in CCR5 expression and CC-chemokine secretion were determined. Migration assays were performed using a 5-microm nitrocellulose filter microchamber system according to the manufacturer's recommendations. Exposure of PBMC to HCV E2 induced a dose-dependent release of regulated on activation normal T-cell-expressed and secreted (RANTES), down-regulation of CCR5 expression and intracellular accumulation of CCR5. This effect was blocked by preincubation of PBMC with anti-CD81. RANTES release following exposure to HCV E2 was mainly attributable to CD8+ cells. After exposure to HCV E2 markedly fewer CD8-positive lymphocytes were attracted by RANTES when compared with CD8+ cells that were studied in the absence of HCV E2. Our results suggest that interaction of HCV E2 with CD81 leads to increased RANTES secretion by CD8+ lymphocytes which induces down-regulation of CCR5 surface via receptor internalization resulting in altered lymphocyte migration.

Effect of Interleukin-2 on Peripheral Blood Mononuclear Cell Cytokine Production and the Hepatic Acute Phase Protein Response

This study investigated the ability of recombinant interleukin-2 (IL-2) to modulate the ability of peripheral blood mononuclear cells (PBMCs) to stimulate an acute phase protein response in isolated human hepatocytes. The effect of IL-2 on the production of tumour necrosis factor-α (TNF) and interleukin-6 (IL-6) by PBMCs isolated from patients with gastrointestinal cancer, multiple organ failure, and healthy controls was also studied. The ability of supernatants from IL-2-treated PBMCs to elicit an acute phase response in hepatocytes was then investigated. IL-2 had no effect on IL-6 or TNF production by PBMCs isolated from any group in the presence or absence of bacterial lipopolysaccharide (LPS). Despite this, preincubation of PBMCs with IL-2 significantly reduced the potential of LPS-stimulated PBMC supernatants to stimulate production of α1 antichymotrypsin, α1-acid glycoprotein, and C-reactive protein by hepatocytes. These observations were not due to a direct effect of IL-2 on hepatocyte acute phase protein production. These findings suggest that in this model IL-2 may modulate PBMC-induced acute phase protein production through an IL-6 and TNF-independent pathway.

Interleukin-18 inhibits human immunodeficiency virus type 1 production in peripheral blood mononuclear cells.

Interleukin (IL)-18 is an interferon (IFN)-gamma-inducing factor and contributes to the Th1 immune response. IL-18 added after infection of peripheral blood mononuclear cells (PBMC) with monocyte-tropic human immunodeficiency virus type 1 (HIV-1) inhibited p24 antigen production by a maximum of 72%. IFN-gamma levels in these cultures were increased, and a significant inverse relationship between HIV-1 production and IFN-gamma levels was observed. A neutralizing anti-IFN-gamma antibody reversed the IL-18 inhibitory effect. Preincubation of PBMC with IL-18 before infection inhibited p24 without additional IL-18 (64%). However, compared with the degree of IL-18 inhibition observed after a 4-day culture, no additional IL-18 inhibitory effect was observed during days 5-13. IL-18 also reduced cell surface expression of the HIV-1 receptor CD4. These results demonstrate that IL-18 inhibited HIV-1 production in PBMC through intermediate IFN-gamma. Furthermore, inhibition was present during the early stages of viral infection and was associated with reduced HIV-1 receptor expression.

In vitro migratory responses of swine neutrophils to actinobacillus pleuropneumoniae.

Swine neutrophils were quantitatively examined for the direct and indirect migratory responses to Actinobacillus pleuropneumoniae (APP) in vitro and the effects of pseudorabies virus (PrV), frequently co-infecting with APP, were also observed. About 30% of swine neutrophils responded to viable APP, while 3.2% of the neutrophils responded to 0.1% casein which served as the control. The migration of APP was not affected by preincubation of neutrophils with PrV, which inhibited the random migration. When the random migration was normalized to 1, the chemotactic indices for APP, opsonized-APP and casein were 64, 70 and 8.5, respectively. Heat-killed APP or E. coli lipopolysaccharide stimulated the production of interleukin-8 activity by adherent peripheral blood mononuclear cells (PBMC). Preincubation of PBMC with PrV inhibited the production of neutrophil attractant activity when stimulated with heat-killed APP. The results suggested that the direct chemotaxis of neutrophils to viable APP might contribute to early infiltration in Actinobacillus pleuropneumonia, and that PrV might inhibit indirect recruitment of neutrophils to infected lungs by compromising the functions of PBMC.

Granulocyte colony-stimulating factor perturbs lymphocyte mitochondrial function and inhibits cell cycle progression

Objective Sera from healthy subjects receiving recombinant human granulocyte colony-stimulating factor (rHuG-CSF) to mobilize CD34 + peripheral blood progenitors (PBPC) have been recently shown to induce unresponsiveness of allogeneic lymphocytes to mitogenic challenge. In the present investigation, the effects of rHuG-CSF on the early stages of lymphocyte activation–induced apoptosis and on lymphocyte cell cycle entry were evaluated. Materials and Methods Sera were obtained from HLA-identical donors receiving rHuG-CSF to mobilize CD34 + PBPC for allogeneic transplantation. Normal peripheral blood mononuclear cells (PBMC) were challenged with phytohemagglutinin (PHA) in the presence of serum collected before (preG) or after rHuG-CSF administration (postG). Mitochondrial function, that is, incorporation of 3,3′-dihexyloxacarbocyanine iodide [DiOC 6(3)] and generation of reactive oxygen species (ROS) as well as expression of c-Myc and Bcl-2 family members (Bcl-2, Bcl-X L, Bax) were evaluated by multiparameter flow cytometry. The activation-induced fragmentation of genomic DNA was detected by highly sensitive LM-PCR assay. Results CD4 +DiOC 6(3) low and CD8 +DiOC 6(3) low T lymphocytes increased and reached 32% (range 27%–38%) and 20% (range 15%–23%) of circulating T cells, respectively, on day 4 of rHuG-CSF administration. Hypergeneration of ROS could be demonstrated in 65% (range 58%–82%) of CD4 + T lymphocytes and in 0.4% (range 0.2%–0.8%) of circulating CD8 + T cells. rHuG-CSF determined no alteration of mitochondrial function if added to allogeneic PBMC in vitro, thus suggesting indirect effects mediated by soluble factors; on the contrary, when PBMC were challenged with PHA in the presence of postG serum, both perturbation of mitochondrial transmembrane potential (Δψ m) and hypergeneration of ROS were induced, and lymphocytes were predominantly arrested in a G 0 -like phase of the cell cycle and displayed genomic DNA fragmentation. Interestingly, the preincubation of PBMC with a blocking antibody directed against CD95 abrogated the perturbation of lymphocyte Δψ m, suggesting that the CD95 signaling pathway might play a role in the induction of apoptosis after PHA stimulation in the presence of postG serum. Moreover, Bax protein was overexpressed in postG ( median fluorescence intensity = 180, range 168–186 ) compared with preG cultures ( median fluorescence intensity = 75, range 68–80; p < 0.01) , while no differences in Bcl-2, Bcl-X L, and c-Myc staining intensity were observed. Conclusions Our findings demonstrate a humoral-mediated rHuG-CSF–induced dissipation of lymphocyte mitochondrial Δψ m; these effects might be mediated by Bax overexpression, with imbalance between apoptosis-promoting and apoptosis-inhibiting Bcl-2 family members and with subsequent induction of mitochondrial permeability transition. Whether immune dysfunction will favorably impact on incidence and severity of acute graft vs host disease after allogeneic PBPC transplantation remains to be determined.

Cytokine-induced modulation of cellular adhesion to human cerebral endothelial cells is mediated by soluble vascular cell adhesion molecule-1.

Tumour necrosis factor-alpha ( TNF-alpha) has been proposed as one of the key mediators of inflammatory diseases of the CNS such as multiple sclerosis. It has been shown to induce the expression of adhesion molecules which is a prerequisite for the transmigration of immune cells through the blood-brain barrier. We therefore investigated the role of TNF-alpha in the expression and release of vascular cell adhesion molecule-1 (VCAM-1) in cultures of human cerebral endothelial cells (HCEC) in comparison with peripheral blood mononuclear cells (PBMC). A time- and dose-dependent expression of VCAM-1 and release of soluble VCAM-1 was detected in HCEC but not PBMC. TNF-alpha-induced release of soluble VCAM-1 was further increased by cotreatment with interferon-beta (IFN-beta), while IFN-beta alone did not affect VCAM-1 expression or the release of soluble VCAM-1. In addition, we observed that preincubation of PBMC with soluble VCAM-1 completely blocked their adhesion to HCEC. In conclusion, the proinflammatory effect of TNF-alpha on HCEC, which involves the induction of VCAM-1 expression and cellular adhesion, is followed by the consecutive effects of soluble VCAM-1 release in blocking adhesion and downregulating further cellular infiltration. Increasing soluble VCAM-1 release during active inflammation could be another mechanism by which IFN-beta treatment exerts protective effects in multiple sclerosis patients.

Nitric Oxide-Mediated Immune Complex-Induced Prostaglandin E2 Production by Peripheral Blood Mononuclear Cells of Humans Infected with Schistosoma mansoni

Granuloma reaction around Schistosoma mansoni eggs is the prominent lesion in human schistosomiasis. Studies have suggested the involvement of a series of suppressive mechanisms in the control of this reaction. Using an in vitro model of granuloma formation, we have shown that immune complexes (IC) isolated from sera of chronic intestinal schistosomiasis patients were able to reduce granulomatous reaction developed against soluble egg antigen-conjugated polyacrylamide beads. In this system, the role of the l-arginine–nitric oxide (NO) pathway in the formation of prostaglandin E2 (PGE2) by human peripheral blood mononuclear cells (PBMC) of patients infected with schistosomiasis was investigated using IC. Preincubation of PBMC with IC produced a significant increase of both nitrite and PGE2 levels in the cell supernatant. This effect was inhibited by coincubation of cells with Nω-nitro-l-arginine methyl ester (L-NAME), a NO synthase inhibitor, showing that the release of PGE2 subsequent to IC stimulation was driven by NO. The inhibitory effect of L-NAME on PGE2 release by IC-treated PBMC was reversed by sodium nitroprusside, a known NO donor. Our results indicate that NO could be an important second signal for the stimulation of PGE2 production induced by IC in PBMC from human schistosomiasis patients.

Effect of a Recombinant Lectin, Viscum album Agglutinin on the Secretion of Interleukin-12 in Cultured Human Peripheral Blood Mononuclear Cells and on NK-Cell-Mediated Cytotoxicity of Rat Splenocytes in vitro and in vivo

A plant lectin, Viscum album agglutinin-I (VAA-I) has been shown to increase the number and cytotoxic activity of natural killer (NK) cells in animal models, but the mechanisms underlying these effects are poorly understood. We investigated the effects of the recombinant form of this lectin (rVAA) on secretion of interleukin (IL)-12 and on NK-mediated cytotoxicity against K562 target cells in cultures of human peripheral blood mononuclear cells (PBMC) as well as against YAC-1 target cells in cultured rat spleen cells. In 24-hour cultures of PBMC, 10 ng/ml plant VAA-I and 50 ng/ml rVAA induced significant increases in the secretion of total IL-12. Its biologically active heterodimeric form, p70, was also significantly induced by rVAA. Preincubation of PBMC or splenocytes for 48 h with rVAA in concentrations ranging between 10 pg/ml and 100 pg/ml resulted in moderate enhancements of NK-mediated cytotoxicity. However, coincubation of a low dose of rVAA (100 pg/ml) together with IL-2 and IL-12 (60 U/ml and 2 U/ml, respectively) led to additive stimulation of NK activity. In in vivo experiments, rVAA showed an enhancing effect on NK activity with a bell-shaped curve of efficacy. Forty-eight hours after a single intravenous injection of its most effective doses, 0.5 and 1 ng/kg, into Wistar rats, the NK cytotoxicity of splenocytes against YAC-1 targets doubled, and the frequency of large granular lymphocytes in peripheral blood showed 2.1- and 3-fold increases as compared to control animals. Twenty-four hours following these low lectin doses, the number of large granular lymphocytes was also significantly elevated. After 48 h, 0.5 ng/kg rVAA induced a significant augmentation in the percentage of peripheral Mac-1+ mononuclear cells, including activated monocytes and NK cells. The present results suggest that rVAA augments the secretion of an active form of IL-12 and potentiates the cytokine-induced NK activation. These effects of rVAA may be related to its stimulatory effects on MHC-unrestricted cytotoxicity in vivo.

Priming of human peripheral blood mononuclear cells with lipopolysaccharides for enhanced arachidonic acid release and leukotriene synthesis.

In a previous study, we have shown that the ability of lipopolysaccharides (LPS) to prime isolated neutrophils for enhanced leukotriene B4 (LTB4) synthesis was dependent on the presence of plasma and involved the CD 14 antigen. In the present study, we have investigated the priming of human peripheral blood mononuclear cells (PBMC) with LPS for the subsequent release and metabolism of arachidonic acid. When PBMC were incubated with LPS for up to 2 h or when freshly isolated PBMC were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or with LPS alone, little or no synthesis of 5-lipoxygenase products nor arachidonic acid liberation were detected. However, the preincubation of PBMC with LPS for as little as 5 min primed cells for the subsequent synthesis of LTB4 upon stimulation with fMLP. Maximal priming was observed following a 15-min preincubation period and the priming effect was transient as cells preincubated with LPS for 90 min or more were no longer primed for leukotriene synthesis. Monocytes were found to be responsible for the enhanced response to fMLP since purified lymphocytes did not produce LTB4 nor LTC4 in contrast to monocyte-enriched suspensions. The priming for leukotriene synthesis coincided with an increased capacity for the release of free arachidonic acid as measured by mass spectrometry; LPS-primed cells released 8-15 times more arachidonic acid than unprimed cells within 1 min of stimulation with fMLP. Priming was observed with as little as 0.001-0.01 microg LPS/mL when cells were incubated in the presence of 10% autologous plasma. Interestingly, in the absence of plasma, priming was only observed at LPS concentrations of 0.1 microg/mL or greater. Pretreatment of cells with anti-CD14 antibodies significantly decreased the priming effect observed with 0.01 microg/mL LPS but did not affect priming with 1 microg/mL LPS. These results indicate that the priming of human PBMC with LPS for the subsequent synthesis of arachidonic acid metabolites via the 5-lipoxygenase pathway is dependent on plasma and CD14 at lower concentrations of LPS (0.001-0.01 microg/mL) but not at LPS concentrations of 0.1 microg/mL or greater.

Stimulation of intercellular adhesion molecule-1 (ICAM-1) antigen expression and shedding by interferon-gamma and phorbol ester in human renal carcinoma cell cultures: relation to peripheral blood mononuclear cell adhesion.

In the present study we investigated the effect of interferon-gamma (IFN-gamma) and phorbol-12-myristate 13 acetate (PMA) on intercellular adhesion molecule-1 (ICAM-1) antigen expression and shedding in human renal carcinoma cell cultures. We also examined the functional consequences of ICAM-1 antigen expression and soluble ICAM-1 molecules on the adhesion of peripheral blood mononuclear cells (PBMC). Incubation of the human renal carcinoma cell line CaKi-1 with IFN-gamma or PMA enhanced ICAM-1 antigen expression. The calcium ionophore, 4-bromo-calcium ionophore A23187 (Bromo-A23187) significantly enhanced the IFN-gamma and PMA effect. Soluble ICAM-1 (sICAM-1) was detected in the supernatants of stimulated but not unstimulated cultures, and correlated significantly with cellular expression. Using 51Cr-labelled peripheral blood mononuclear cells in a cell adhesion assay, we demonstrated increased adhesion in IFN-gamma-treated CaKi-1 cultures, which was augmented by Bromo-A23187. This adhesion was blocked by preincubation of CaKi-1 cells with monoclonal antibody against ICAM-1 or by preincubation of PBMC with either monoclonal antibody against leucocyte function associated antigen-1 alpha (LFA-1 alpha), a major receptor for ICAM-1, supernatants from treated cultures or purified sICAM-1 molecules. Thus, shedding of ICAM-1 may play a role during the escape from immunosurveillance by renal carcinoma cells.