ARTHRITIS & RHEUMATISM, Issue 5 2003
Joel A. G. Van Roon
Objective To determine whether monocyte/macrophages from rheumatoid arthritis (RA) patients can be selectively eliminated by a toxin-conjugated antibody CD64,ricin A (CD64-RiA) directed toward the high-affinity receptor for IgG (Fc,RI), exploiting the capacity of Fc,RI to efficiently endocytose antibody which it has bound. Methods Mononuclear cells from peripheral blood (PB) and synovial fluid (SF) obtained from RA patients were cultured in the presence of CD64-RiA. Cell death of monocyte/macrophages was measured by phenotypic changes (light-scatter patterns and CD14 and Fc,RI expression) and apoptosis (nuclear DNA fragmentation). We then tested whether CD64-RiA,induced cell death of macrophages affected their capacity to stimulate antigen-induced lymphocyte proliferation and to secrete cytokines. Additionally, the capacity of CD64-RiA to inhibit proinflammatory activity and cartilage degradation by RA synovial tissue explants was evaluated. Results Inflammatory macrophages from RA SF expressed elevated levels of Fc,RI and were selectively eliminated by CD64-RiA via apoptotic cell death. Monocyte/macrophages from RA PB, which had lower levels of Fc,RI expression, were much less affected. Induction of SF macrophage apoptosis was associated with efficient inhibition of antigen-induced lymphocyte proliferation and a reduction in tumor necrosis factor , (TNF,) release. Consistent with these effects on SF macrophages, CD64-RiA also inhibited TNF, production, interleukin-1, production, and cartilage-degrading activity of RA synovial tissue explants. Conclusion Together, these data underscore the crucial role of synovial macrophages in RA joint inflammation and indicate that selective elimination of these cells through Fc,RI-directed immunotoxins could be a novel approach to the treatment of RA. [source]

Human Immunodeficiency Virus Infection of the Brain: Pitfalls in Evaluating Infected/Affected Cell Populations

BRAIN PATHOLOGY, Issue 1 2004
Stephanie J. Bissel
Monocyte/macrophages and CD4 T-cells are the primary hematopoietic targets of productive HIV infection. In the brain, potential cellular targets for HIV infection include perivascular and parenchymal macrophages/microglia, oligodendrocytes, endothelia, neurons, and astrocytes. We examine evidence of productive and non-productive infection for each cell type in the brains of HIV-infected patients with and without HIV encephalitis. Despite the voluminous literature and substantial experimental effort over the past two decades, evidence for productive infection of any brain cell other than macrophages is left wanting. [source]

Monocytes and T lymphocytes contribute to a predominance of interleukin 6 and interleukin 10 in systemic lupus erythematosus,

CYTOMETRY, Issue 4 2009
Susana Mellor-Pita
Abstract Objective To investigate the contribution of T lymphocytes and monocytes to cytokine production in systemic lupus erythematosus (SLE). Methods Forty-five SLE patients and 19 healthy volunteers were included. Serum levels of tumor necrosis factor alpha (TNF,), interferon gamma (IFN,), interleukin (IL)-6, and IL10 were quantified by ELISA. The cytokine production capacities of peripheral blood mononuclear cells were assessed by culturing in vitro with PMA+Ionomycin or LPS. The intracellular cytokine expression was measured by flow cytometry in T lymphocytes and monocytes, respectively. The influence of the disease activity (measured as the SLE-disease activity index; SLEDAI) and the treatment the patients were receiving was evaluated. Results Serum IL10, IL6, and TNF, levels were increased in patients (P , 0.01), and a higher spontaneous (without stimuli) intracellular expression of IL10 in CD4+ and CD8+ T lymphocytes (P < 0.05) and of IL6 in monocytes (P = 0.01) were found. After stimulation, patients presented a higher percentage of CD4+ and CD8+ T lymphocytes producing IL4 and IL10 (P , 0.01), and of monocytes producing IL6 (P = 0.04) and IL10 (P = 0.008). The SLEDAI score was positively correlated with the percentage of CD4+IL10+ and CD8+IL10+ T lymphocytes (P < 0.01), and inversely correlated with CD8+TNF,+ (P= 0.02), CD4+IFN,+ (P = 0.04) and CD8+ IFN,+ (P = 0.002) T lymphocytes. Patients receiving high dose prednisone produced higher IL10, but they also were the patients with a more active disease. Conclusion Monocytes and T lymphocytes (CD4+ and CD8+) contribute to an overproduction of IL6 and IL10 in SLE; this correlates with the disease activity but is independent of the treatment the patients are receiving. © 2009 Clinical Cytometry Society [source]

Rapid infusion of a phospholipid emulsion attenuates the effects of endotoxaemia in horses

EQUINE VETERINARY JOURNAL, Issue 3 2007
J. N. MOORE
Summary Reasons for performing study: Endotoxaemia currently is associated with a poor prognosis in horses. The results of recent trials in other species indicate that phospholipid emulsions reduce the deleterious effects of endotoxin (LPS). However, in a previous study in horses, a 2 h infusion of emulsion caused an unacceptable degree of haemolysis. Hypothesis: Rapid administration of a lower total dose of emulsion would reduce the effects of LPS and induce less haemolysis; the emulsion would reduce inflammatory effects of LPS in vitro. Methods: Twelve healthy horses received an i.v. infusion either of saline or a phospholipid emulsion (100 mg/kg), followed immediately by E. coli O55:B5 LPS (30 ng/kg). Clinical parameters, haematological profiles, serum tumour necrosis factor (TNF) activity, serum lipid profiles, urine analyses and severity of haemolysis were monitored before and at selected times after LPS. Monocytes were also incubated in vitro with LPS in the presence or absence of emulsion, after which TNF and tissue factor activities were determined. Results: Clinical signs of endotoxaemia were reduced in horses receiving the emulsion, including clinical score, heart rate, rectal temperature, serum TNF activity, and the characteristic leucopenic response to LPS, when compared to horses not receiving the emulsion. Three horses receiving the emulsion had none, 2 had mild and one had moderate haemolysis. There were no differences in urinalysis results and creatinine concentrations, either within the groups over time or between the groups. Serum concentrations of phosphatidylcholine, bile acids and triglycerides peaked immediately after the infusion; there were no significant changes in concentrations of nonesterified fatty acids or cholesterol. Incubation of equine monocytes with emulsion prevented LPS-induced TNF and tissue factor activities. Conclusions: Rapid administration of emulsion significantly reduced inflammatory effects of LPS in vivo and caused a clinically insignificant degree of haemolysis. The results of the in vitro studies indicate that emulsion prevents not only LPS-induced synthesis of cytokines, but also expression of membrane-associated mediators (i.e. tissue factor). Potential relevance: Rapid i.v. administration of emulsions containing phospholipids that bind endotoxin may provide a clinically useful method of treating endotoxaemia in horses. [source]

Procoagulant and inflammatory response of virus-infected monocytes

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 10 2002
J. J. M. Bouwman
Abstract Background Monocytes play a prominent role in inflammation, coagulation and atherosclerosis by their ability to produce tissue factor (TF) and cytokines. The aim of the present study was to establish whether virus-infected monocytes initiate coagulation. In addition, the production of cytokines by monocytes may accelerate the chronic process of atherosclerosis and may contribute to coronary syndromes by eliciting plaque instability. Materials and methods Monocytes were isolated by Vacutainer®, BD Biosciences, Alphen aan den Rijn, Netherlands and subsequent magnetic cell sorting (MACS®, Milteny Biotec, Bergish Gladbach, Germany). Coagulation times in normal pooled plasma and Factor VII-deficient plasma were measured after infection with cytomegalovirus (CMV), Chlamydia pneumoniae (Cp) and influenza A\H1N1. Anti-TF antibodies were added to neutralize TF expressed on monocytes. Interleukins (IL) 6, 8 and 10 were measured in the supernatants. Results Chlamydia pneumoniae- and CMV-infected monocytes decreased the clotting time by 60%, and influenza-infected monocytes by 19%, as compared to uninfected monocytes. Procoagulant activity was absent when Factor VII-deficient plasma or anti-TF antibodies were used. Monocytes produced both IL-6 and IL-8 after infection with CMV (317 pg mL,1 and 250 pg mL,1) or Cp (733 pg mL,1 and 268 pg mL,1). Similar results were obtained for influenza virus-infected monocytes, but the levels of both cytokines were 3,5-fold higher (1797 pg mL,1 and 725 pg mL,1). Interleukin-10 was not produced by infected monocytes. Conclusion The procoagulant activity of virus-infected monocytes is TF-dependent. Although influenza infection did not generate a significant reduction in clotting time, the pronounced expression of IL-6 and IL-8 may induce local and/or systemic inflammatory reactions, which may be associated with plaque rupture and atherosclerosis. The lack of production of the anti-inflammatory cytokine IL-10 may even accelerate these processes. [source]

Systemic increase in type,I interferon activity in Sjögren's syndrome: A putative role for plasmacytoid dendritic cells

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2008
Manon
Abstract In the salivary glands of primary Sjögren's syndrome (pSjS) patients, type,I IFN activity is increased, but systemic levels of type,I IFN proteins are rarely detected. This study focused on the systemic activity of type,I IFN in pSjS, as well as the role of peripheral plasmacytoid dendritic cells (pDC). Monocytes obtained from pSjS patients showed an increased expression of 40,genes. Twenty-three of these genes (58%), including IFI27, IFITM1, IFIT3 and IFI44, were inducible by type,I IFN. pSjS serum had an enhanced capability of inducing IFI27, IFITM1, IFIT3 and IFI44 in the monocytic cell line THP-1, likely due to the action of IFN-,. This effect could be inhibited by blocking the type,I IFN receptor, supporting a high type,I IFN bioactivity in pSjS serum. In addition, circulatory pDC showed increased expression of CD40. This expression was correlated to the expression level of the type,I IFN-regulated genes IFI27 and IFITM1 in monocytes of the same individual. This study indicates that the increased type,I IFN activity observed in pSjS patients is not only a local but also a systemic phenomenon and points to pDC as a possible source of this activity. [source]

IL-15-induced human DC efficiently prime melanoma-specific naive CD8+ T cells to differentiate into CTL

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2007
Peter Dubsky
Abstract Monocytes differentiate into dendritic cells (DC) in response to GM-CSF combined with other cytokines including IL-4 and IL-15. Here, we show that IL15-DC are efficient in priming naive CD8+ T cells to differentiate into melanoma antigen-specific cytotoxic T,lymphocytes (CTL). While both melanoma peptide-pulsed IL15-DC and IL4-DC expand high-precursor frequency MART-1-specific CD8+ T cells after two stimulations in vitro, IL15-DC require much lower peptide concentration for priming. IL15-DC are more efficient in expanding gp100-specific CD8+ T cells and can expand CD8+ T cells specific for Tyrosinase and MAGE-3. CTL primed by IL15-DC are superior in their function as demonstrated by (i),higher IFN-, secretion, (ii),higher expression of Granzyme,B and Perforin, and (iii),higher killing of allogeneic melanoma cell lines, most particularly the HLA-A*0201+ Sk-Mel-24 melanoma cells that are resistant to killing by CD8+ T cells primed with IL4-DC. Supernatants of the sonicated cells demonstrate unique expression of IL-1, IL-8 and IL-15. Therefore, membrane-bound IL-15 might contribute to enhanced priming by IL15-DC. Thus, IL-15 induces myeloid DC that are efficient in priming and maturation of melanoma antigen-specific CTL. [source]

Monocytes in the rat: Phenotype and function during acute allograft rejection

IMMUNOLOGICAL REVIEWS, Issue 1 2001
Birte Steiniger
Summary: Cells of the monocyte/macrophage system originate from the bone marrow, reach the organs via the blood, immigrate through post-capillary venules and further differentiate into organ-specific tissue macrophages. In rats and other species, activated monocytes/macrophages aggravate autoimmune reactions, rejection of non-vascularized allografts and chronic allograft rejection. It is very likely that they also contribute to acute allograft destruction. So far it has been impossible to distinguish the function of monocytes from that of macrophages, because cell phenotypes and their alterations upon activation are ill-defined. We have thus begun to characterize the ex vivo phenotype and function of rat monocytes in the normal state and during renal allograft rejection. Monocytes are recovered from both the central and the marginal blood pool by perfusing either the recipient's circulation or the allograft vasculature. Rat monocytes have a unique surface phenotype. During allograft rejection or after infusion of interferon-, they up-regulate class II MHC molecules, CD161 (NKR-P1A), CD62L and CD8, while CD4 and CD43 are down-modulated. Activated perfusate monocytes exert increased in vitro cytotoxicity against tumour targets, which differs from that of NK cells. We speculate that activated monocytes contribute to kidney allograft destruction by directly damaging endothelial cells or by promoting intravascular coagulation. [source]

Expression of GM1, a marker of lipid rafts, defines two subsets of human monocytes with differential endocytic capacity and lipopolysaccharide responsiveness

IMMUNOLOGY, Issue 4 2007
M. Maximina Bertha Moreno-Altamirano
Summary Monocytes constitute 5,10% of total human peripheral blood leucocytes and remain in circulation for several days before replenishing the tissue macrophage populations. Monocytes display heterogeneity in size, granularity and nuclear morphology, and in the expression of cell membrane molecules, such as CD14, CD16, CD32, CD64, major histocompatibility complex class II, CCR2, CCR5, among others. This has led to the suggestion that individual monocyte/macrophage populations have specialized functions within their microenvironments. This study provides evidence for the occurrence of two peripheral blood monocyte subpopulations on the basis of their differential expression of GM1, a sphingolipid found mostly in lipid rafts, a CD14+ GM1low population and a CD14+ GM1high population comprising about 97·5% and 2·5% of total CD14+ cells, respectively. GM1 expression correlates with functional differences in terms of endocytic activity, susceptibility to mycobacterial infection, and response to lipopolysaccharide (LPS) (modulation of Toll-like receptor-4 expression). CD14+ GM1low cells proved to be less endocytic and more responsive to LPS, whereas CD14+ GM1high cells are more endocytic and less responsive to LPS. In addition, during monocyte to macrophage differentiation in vitro, the percentage of CD14+ GM1high cells increases from about 2·5% at day 1 to more than 50% at day 7 of culture. These results suggest that GM1low and GM1high monocytes in peripheral blood, represent either different stages of maturation or different subsets with specialized activities. The expression of CD16 on GM1high favours the first possibility and, on the other hand that up-regulation of GM1 expression and probably lipid rafts function is involved in the monocyte to macrophage differentiation process. [source]

Expression of ,-defensin 1 and 2 mRNA by human monocytes, macrophages and dendritic cells

IMMUNOLOGY, Issue 4 2002
Louise A. Duits
Summary Human ,-defensins are broad-spectrum antimicrobial peptides known to be produced by epithelial cells. It was recently shown that ,-defensins also display chemotactic activity for dendritic cells (DC) and T cells, and thus may serve to link innate and adaptive immunity. The aim of the present study was to explore expression of mRNA for these peptides in mononuclear phagocytes and DC. The results revealed that monocytes, monocyte-derived-macrophages (MDM), and monocyte-derived-dendritic cells (DC) all express human-,-defensin-1 (hBD-1) mRNA. hBD-1 mRNA expression by monocytes and MDM was increased after activation with interferon-, (IFN-,) and/or lipopolysaccharide (LPS) in a dose- and time-dependent fashion. Alveolar macrophages showed an intense hBD-1 expression, which could not be further increased. Expression of hBD-1 mRNA by immature DC was low, and increased considerably after maturation. Monocytes, MDM, alveolar macrophages and DC showed a limited expression of human ,-defensin-2 (hBD-2) mRNA, which could only be increased in monocytes and alveolar macrophages by IFN-, and/or LPS in a dose- and time-dependent fashion. Immunocytochemical stainings demonstrated the expression of hBD-2 peptide by freshly isolated blood monocytes and alveolar macrophages in cytospin preparations. [source]

NOD2/CARD15 genotype influences MDP-induced cytokine release and basal IL-12p40 levels in primary isolated peripheral blood monocytes

INFLAMMATORY BOWEL DISEASES, Issue 8 2008
Vanessa Beynon MD
Abstract Background: The functional link between mutations in NOD2 and Crohn's disease (CD) has not been entirely elucidated. The 1007fs mutation results in loss of NF-,B activation in response to muramyl dipeptide (MDP) but has also been linked to an increased IL-1, processing and IL-12 release. Methods: We investigated the basal and MDP-triggered mRNA expression and protein release for TNF-,, IL-10, IL-1,, and IL-12p40 in peripheral blood monocytes from 40 CD patients and 15 healthy individuals with different NOD2 genotypes. Results: Monocytes from individuals with 2 mutated NOD2 alleles (homozygous and compound-heterozygous individuals) displayed an impaired release of TNF-, and IL-10 but also of IL-1, and IL-12p40 in response to MDP. In contrast to other NOD2 variants, the presence of at least 1 1007fs allele in double-mutated individuals completely abrogated NOD2 receptor function. Interestingly, monocytes from CD patients with 2 mutated NOD2 alleles displayed significantly higher basal levels of IL-12p40 in cell culture supernatants compared to wildtype CD patients and control individuals (P = 0.002 and P = 0.008, respectively). This was regardless of the IL23R genotype and was not mirrored by increased IL-12p40 plasma levels in these individuals. Conclusions: The CD-associated NOD2 variants lead, in a dose- and mutation-dependent manner, to an impaired release of TNF-,, IL-10, IL-1,, and IL-12p40 in response to MDP. The finding of increased basal levels for IL-12p40-related cytokines in monocytes with 2 mutated NOD2 alleles is likely to set a new link between NOD2 mutations and the inflammatory mechanisms underlying CD. (Inflamm Bowel Dis 2008) [source]

Activated monocytes and platelet-monocyte aggregates in patients with sickle cell disease*

INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 2 2002
TED WUN
Tumour necrosis factor-, (TNF-,) and interleukin-1, (IL-1,) increase endothelial surface receptors that mediate the adherence of sickle erythrocytes to the endothelium. Increased circulating levels of these cytokines have been found in patients with sickle cell disease (SCD). Monocytes are a source of both of these inflammatory mediators; we therefore determined whether circulating monocytes were activated in SCD, as defined by intracellular expression of these cytokines. Blood was also assayed for the presence of platelet,monocyte aggregates (PMAs), as platelet adherence is one possible mechanism for monocyte activation. The median percentages of monocytes expressing intracellular TNF-, and IL-1, in SCD patients were 6.8 (2.8,17.3) [median (range)] and 14.1 (1.3,44.8), respectively. In African-American controls the corresponding percentages were 0.3 (0.1,0.5) and 0.4 (0.1,3.0), and in Caucasians 0.2 (0.1,0.5) and 0.8 (0.8,1.9) (P < 0.001, Kruskal,Wallis). The mean percentage (± SD) of PMA was 14.0 ± 8.3 for Caucasian controls, 25.7 ± 7.3 for African-American controls, and 45.7 ± 21.6 for patients with SCD (P < 0.001, RM ANOVA; P < 0.05, Newman,Keuls posthoc test). We conclude that there are increased circulating PMAs and monocyte activation in patients with SCD. [source]

Gene Expression Profiling in Paget's Disease of Bone: Upregulation of Interferon Signaling Pathways in Pagetic Monocytes and Lymphocytes,,§

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2008
Zsolt B Nagy
Abstract We examined the gene expression profile of genes involved in bone metabolism in 23 patients with PD compared with 23 healthy controls. We found a significant overexpression of the genes of the IFN pathway along with a downregulation of tnf-,. Our result suggest that IFN-mediated signaling may play important roles in aberrant osteoclastogenesis of PD. Introduction: Paget's disease of bone (PD) is characterized by focal regions of highly exaggerated bone remodeling and aberrant osteoclastogenesis. Under physiological conditions, circulating monocytes may serve as early progenitors of osteoclasts and along with peripheral blood lymphocytes produce a wide variety of factors important in bone metabolism. Nevertheless, little is known about the roles of circulating monocytes and lymphocytes in relation to the pathological bone turnover in PD. Materials and Methods: In this study, we aimed at investigating the gene expression pattern of PD using quantitative real-time PCR in monocytes and lymphocytes isolated from peripheral blood mononuclear cells (PBMCs). Fifteen genes known to be involved in osteoclastogenesis were studied in cells from 23 patients with PD and in cells from 23 healthy controls. Eight human genes including ifn-, (3.48-fold, p < 0.001), ifn-, (2.68-fold, p < 0.001), ifn-, (1.98-fold, p = 0.002), p38 ,2 mapk (2.47-fold, p = 0.002), ifn-,r1 (2.03-fold, p = 0.01), ifn-,r2 (1.81-fold, p = 0.02), stat1 (1.57-fold, p = 0.037), and tnf-, (,2.34, p < 0.001) were found to be significantly altered in pagetic monocytes compared with monocytes of healthy controls. Results: In pagetic lymphocytes, significant changes in the expression of ifn-, (2.17-fold, p < 0.001), ifn-, (2.13-fold, p = 0.005), ifn-, (1.89-fold, p < 0.001), ifn-,r1 (1.02-fold, p = 0.04), ifn-,r2 (1.01-fold, p = 0.031), stat2 (1.79-fold, p < 0.001), and tnf-, (,1.49, p < 0.001) were found compared with lymphocytes of healthy controls. Furthermore, IFN-, protein was significantly elevated in the sera of PD patients (18.7 ± 6.69 pg/ml) compared with healthy controls (3.87 ± 6.48 pg/ml, p = 0.042). Conclusions: In conclusion, our data suggest that novel pathways mainly related to the IFN-mediated signaling may play important roles in the aberrant osteoclastogenesis of PD. [source]

Combination of Cobe AutoPBSC and Gambro Elutra as a platform for monocyte enrichment in dendritic cell (DC) therapy: Clinical study

JOURNAL OF CLINICAL APHERESIS, Issue 5 2008
Ying Chen
Abstract Monocytes are a common source for generating dendritic cells (DCs). The aim of the present study was to evaluate the efficiency of a platform for monocyte collection and enrichment in a clinical setting. The platform was based on the combination of two semiautomated devices; the Cobe Spectra Auto PBSC for mononuclear cells (MNC) collection followed by counterflow elutriation for monocyte enrichment (Gambro BCT Elutra). Twenty-four patients with various types of epithelial cancer participated in the study. MNC collections were first performed as large volume leukapheresis (LVL). Subsequently, MNC products were processed with an elutriation system for monocyte isolation. LVL resulted in the collection of MNC at a median of 8.1 × 109 cells, containing of 31.4% monocytes. A similar efficacy was also shown in patients with lower peripheral blood counts. Elutriation of the MNC product with the Cobe Elutra device resulted in the enrichment of monocytes at a median of 2.7 × 109 cells, with a recovery of 80.2% and a purity of 90.7%. These monocytes were then successfully developed into DCs for clinical therapy after in vitro manipulation. These data suggest that the combination of the Cobe Spectra Auto PBSC and the Gambro BCT Elutra is an effective platform for monocyte enrichment in clinical practice according to GCP standards and GMP guidelines, and can be easily implemented in the clinical routine under current DC protocols. J. Clin. Apheresis, 2008. © 2008 Wiley-Liss, Inc. [source]

Closed system generation of dendritic cells from a single blood volume for clinical application in immunotherapy,

JOURNAL OF CLINICAL APHERESIS, Issue 4 2005
M. Elias
Dendritic cells (DC) used for clinical trials should be processed on a large scale conforming to current good manufacturing practice (cGMP) guidelines. The aim of this study was to develop a protocol for clinical grade generation of immature DC in a closed-system. Aphereses were performed with the Cobe SpectraÔ continuous flow cell separator and material was derived from one volume of blood processed. Optimisation of a 3-phase collection autoPBSC technique significantly improved the quality of the initial mononuclear cell (MNC) product. Monocytes were then enriched from MNC by immunomagnetic depletion of CD19+ B cells and CD2+ T cells and partial depletion of NK cells using the Isolex 300I Magnetic cell selector. The quality of the initial mononuclear cell product was found to determine the outcome of monocyte enrichment. Enriched monocytes were cultured in Opticyte gas-permeable containers using CellGro serum-free medium supplemented with GM-CSF and IL-4 to generate immature DC. A seeding concentration of 1 × 106 was found optimal in terms of DC phenotype expression, monocyte percentage in culture, and cell viability. The differentiation pattern favours day 7 for harvest of immature DC. DC recovery, viability, as well as phenotype expression after cryopreservation of immature DC was considered in this study. DC were induced to maturation and evaluated in FACS analysis for phenotype expression and proliferation assays. Mature DC were able to generate an allogeneic T-cell response as well as an anti-CMV response as detected by proliferation assays. These data indicate that the described large-scale GMP-compatible system results in the generation of stable DC derived from one volume of blood processed, which are qualitatively and quantitatively sufficient for clinical application in immunotherapeutic protocols. J. Clin. Apheresis © 2005 Wiley-Liss, Inc. [source]

Inhibitory effects of N -acetylcysteine on scavenger receptor class A expression in human macrophages

JOURNAL OF INTERNAL MEDICINE, Issue 5 2002
L. SVENSSON
Abstract.,Svensson L, Norén K, Wiklund O, Lindmark H, Ohlsson B, Mattsson Hultén L (Wallenberg Laboratory for Cardiovascular Research, The Sahlgrenska Academy at Göteborg University, Göteborg; and AstraZeneca, Mölndal, Sweden). Inhibitory effects of N -acetylcysteine on scavenger receptor class A expression in human macrophages. J Intern Med 2002; 251:. Objective.,The formation of foam cells from monocyte-derived macrophages involves the uptake of modified lipoproteins by scavenger receptors. Antioxidants inhibit lipoprotein oxidation and may also modulate gene expression. We investigated the effect of the antioxidant N -acetylcysteine on the expression of the class A scavenger receptor (SR-A) types I and II in human macrophages. Design.,Monocytes and macrophages from healthy blood donors and plaque-derived macrophages from patients undergoing carotid endartherectomy were used for experiments. SR-A mRNA was analysed with quantitative and semiquantitative reverse transcription-polymerase chain reaction, and ligand binding and uptake were assessed with 125I-labelled acetylated low-density lipoprotein (LDL). Results.,Incubation of monocytes and monocyte-derived macrophages with N -acetylcysteine decreased both SR-A I and II mRNA expression. N -Acetylcysteine also reduced SR-A mRNA in lesion-derived cells. Binding and uptake of 125I-acetylated LDL was decreased after brief incubation with N -acetylcysteine. After longer periods of incubation with N -acetylcysteine we observed an increased degradation of lipoproteins. Conclusions.,Our results imply that N -acetylcysteine leads to a decrease in SR-A mRNA and initially also to an attenuated uptake of modified lipoproteins. This adds more to the knowledge about the cellular actions of this drug. [source]

Suppression of proinflammatory cytokines and induction of IL-10 in human monocytes after coxsackievirus B3 infection

JOURNAL OF MEDICAL VIROLOGY, Issue 4 2001
P. Hofmann
Abstract Coxsackievirus B3 (CVB3) causes acute and chronic myocarditis, which is accompanied by an intense mononuclear leukocyte infiltration. Because myocardial tissue damage may either result from viral infections or from a dysregulated immune response, the susceptibility of human monocytes and macrophages to CVB3 was examined in this study with regard to virus replication, virus persistence, and release of cytokines. Monocytes were infected by CVB3 as shown by the intracellular appearance of plus- and minus-strand viral RNA, which was also capable of persisting for more than 10 days. Fresh monocytes were not permissive for full virus replication whereas monocyte-derived macrophages yielded a low amount of new viruses, which led to cell death. Although CVB3 infection induced the mRNA for the proinflammatory cytokines tumor necrosis factor-alpha (TNF-,), interleukin (IL)-1, and IL-6, only little cytokine production occurred. When infected monocytes were stimulated in addition by lipopolysaccharides (LPS), cytokine production was partially suppressed. In striking contrast, IL-10 expression was strongly and persistently induced by CVB3 on the mRNA and the protein level. These data show a dysregulated cytokine response in CVB3-exposed human monocytes and macrophages, which is characterized by a suppression of proinflammatory cytokines and a dominance of IL-10. This viral strategy may aid CVB3, causing chronic myocardiopathy. J. Med. Virol. 64:487,498, 2001. © 2001 Wiley-Liss, Inc. [source]

Transfusion in premature infants impairs production and/or release of red blood cells, white blood cells and platelets

JOURNAL OF PAEDIATRICS AND CHILD HEALTH, Issue 3 2002
B Frey
Objective: To examine whether red blood cell transfusion in infants with anaemia of prematurity alters peripheral counts of red blood cell precursors, total white blood cells and white cell differential and platelets. Methodology: In 18 consecutive stable premature infants with anaemia of prematurity, peripheral cell counts were prospectively recorded immediately before transfusion of 20 mL/kg packed red blood cells (given over 6 h), and at 48 h after completion of the transfusion. Results: The median (interquartile range) haematocrit increased from 22.0% (21.3,24.0%) pre-transfusion to 37.0% (36.0,38.0%) post-transfusion (P < 0.001). Red-cell precursors decreased: median (interquartile range) reticulocytes from 3.7% (3.0,7.7%) to 3.7% (2.6,4.1%) (P = 0.03); and median (interquartile range) nucleated red blood cells from 0 G/L (0,0.2 G/L) to 0 G/L (0,0 G/L) (P = 0.03). The mean (SD) platelet count decreased from 420 G/L (154 G/L) to 313 G/L (101 G/L) (P = 0.001). The total white blood cell count and neutrophils did not change significantly; however, median (interquartile range) immature neutrophils decreased from 0.12 G/L (0.06,0.74 G/L) to 0.08 G/L (0.01,0.24 G/L) (P = 0.03). Lymphocytes, eosinophils, basophils and plasma cells remained unchanged. Monocytes increased (P = 0.01). Conclusions: Forty-eight hours after red blood cell transfusion to premature infants, there is an absolute decrease in red blood cell precursors, immature white blood cells and platelets, probably due to erythropoietin-suppression. [source]

Alcohol Exposure Impairs Myeloid Dendritic Cell Function in Rhesus Macaques

ALCOHOLISM, Issue 9 2009
Robert W. Siggins
Background:, Alcohol intoxication suppresses both the innate and adaptive immunities. Dendritic cells (DCs) are the major cell type bridging the innate and acquired immune responses. At the present time, the effects of alcohol on DC development in hematopoietic tissues and the functional activities of DCs are incompletely elucidated. This study investigated the impact of chronic alcohol exposure on the alteration of hematopoietic precursor cell and DC populations in the bone marrow and peripheral blood of rhesus macaques. Methods:, Rhesus macaques were administered alcohol or isocaloric sucrose daily for a period of 3 months through surgically implanted gastric catheters. Peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs) were isolated for flow cytometric analysis after 3 months. Monocytes were cultured with human IL-4 (10 ng/ml) and GM-CSF (50 ng/ml) in the absence and presence of alcohol (50 mM). On day 6 of the culture, a cocktail of stimulants including IL-1, (18 ng), IL-6 (1800 U), TNF-, (18 ng), and PGE2 (1.8 ,g) were added to the designated wells for transformation of immature dendritic cells (iDCs) to mature myeloid DCs. The cells were analyzed on day 8 by flow cytometry for expression of DC costimulatory molecule expression. Results:, EtOH-treated animals had significantly lower numbers of myeloid DCs (lineage-HLA-DR+CD11c+CD123,) in both the PBMCs and BMCs compared to controls (5,654 ± 1,273/106 vs. 2,353 ± 660/106 PBMCs and 503 ± 34 vs. 195 ± 44/106 BMCs). Under culture conditions, the number of lineage-HLA-DR+CD83+ cells was low in control wells (0.38 ± 0.08%). Alcohol inhibited the increase in the number of lineage-HLA-DR+CD83+ cells in iDC wells (2.30 ± 0.79% vs. 5.73 ± 1.40%). Alcohol also inhibited the increase in the number of lineage-HLA-DR+CD83+ cells in mature DC wells (1.23 ± 0.15% vs. 4.13 ± 0.62%). Conclusions:, Chronic EtOH decreases the bone marrow and circulating pools of myeloid DCs. Additionally, EtOH suppresses costimulatory molecule CD83 expression during DC transformation, which may attenuate the ability of DCs to initiate T-cell expansion. [source]

Alcohol-Induced Up-Regulation of Fibrinolytic Activity and Plasminogen Activators in Human Monocytes

ALCOHOLISM, Issue 8 2002
Edlue M. Tabengwa
Background Moderate alcohol consumption is associated with reduced risk for coronary heart disease. This may due, in part to increased fibrinolysis. Monocytes synthesize fibrinolytic proteins, tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and their receptors. These studies were carried out to determine the effect of low alcohol on monocyte fibrinolytic activity and PA messenger RNA (mRNA) synthesis. Methods Peripheral blood monocytes and U937 cells were incubated in absence/presence of low alcohol (0.1%, v/v) for various times (0,1 hr), followed by incubations in the absence of alcohol (0,24 hr) before measurement of fibrinolytic activity and PA mRNA levels (reverse transcriptase polymerase chain reaction). Results Brief exposure (15 min, 4°C) of U937 cells to low alcohol resulted in an approximately 2- to 3-fold increase (269.0 ± 5.6 fmol/1 × 106 cells versus 656.0 ± 94.0 fmol/1 × 106 cells) in fibrinolytic activity. Preincubation of U937 cells and peripheral blood monocytes in low alcohol (1 hr, 37°C) followed by incubation in the absence of alcohol (24 hr) resulted in a sustained approximately 4- to 5-fold increase (414.0 ± 174.7 vs. 965.33.0 ± 104.8 fmol/1 × 106 cells) and an approximately 3- to 4-fold (20.5 ± 2.14 vs. 74 ± 2.28 fmol/2 × 106 cells, respectively) increase in fibrinolytic activity. Preincubation of monocytes with low alcohol (1 hr, 37°C) followed by incubation in the absence of alcohol (6 hr) resulted in an approximately 5- to 6-fold (0.06 ± 0.02 vs. 0.42 ± 0.02) and an approximately 2- to 3-fold (0.89 ± 0.04 vs. 2.07 ± 0.29) increase in t-PA and u-PA mRNA (reverse transcriptase polymerase chain reaction; PA/glyceraldehyde-3-phosphate dehydrogenase ratio), respectively. Conclusions These data suggest that low alcohol exerts a rapid, direct, and sustained effect on monocyte fibrinolytic activity, which may be, due in part, to increased monocyte t-PA/u-PA expression. These data provide a feasible molecular mechanism by which alcohol effects on monocyte fibrinolysis may contribute to the cardioprotective benefit associated with moderate alcohol consumption. [source]

Fibronectin-adherent monocytes express tissue factor and tissue factor pathway inhibitor whereas endotoxin-stimulated monocytes primarily express tissue factor: physiologic and pathologic implications

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2007
M. S. BAJAJ
Summary Background:,Monocytes are critical cells in initiating physiologic and/or pathologic tissue factor (TF)-induced intravascular and extravascular coagulation. Monocytes constitutively express small amounts of TF and tissue factor pathway inhibitor (TFPI). Non-adherent lipopolysaccharide (LPS)-stimulated monocytes express significant amounts of TF; however, increased expression of TFPI by these cells is controversial. Further, whether fibronectin-adherent monocytes (mimicking conditions in the extravascular space) express sufficient TFPI to inhibit TF-procoagulant activity (PCA) is unknown.Objective:,To compare TF and TFPI expression by fibronectin-adherent and LPS-stimulated non-adherent monocytes.Methods:,Monocytes were isolated from normal peripheral blood, adhered to fibronectin or stimulated with lipopolysaccharide (LPS) under non-adherent conditions and examined for expression of TF and TFPI using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), ELISA and factor X (FX) activation.Results:,Under LPS-free conditions, the fibronectin-adherent monocyte TF mRNA, antigen and activity were markedly upregulated. Notably, cell and microparticle (MP)-associated TF and alternatively spliced TF (asTF) were all upregulated. TFPI mRNA and antigen were also upregulated in the fibronectin-adherent monocytes, which significantly inhibited TF-PCA. TFPI mRNAs for both alpha and beta forms were detected. The peak in TFPI activity occurred in tandem with the peak in TF-PCA. In contrast, LPS-stimulated monocytes, which expressed cell and MP-associated TF and asTF, demonstrated only minimal expression of TFPI as determined by mRNA, antigen or inhibition of TF activity.Conclusion:,Both LPS-stimulated and fibronectin-adherent monocytes demonstrate a procoagulant phenotype by expressing TF but only fibronectin-adherent monocytes express significant amounts of TFPI to control thrombin generation and fibrin formation in the context of extravascular space. [source]

Actinobacillus actinomycetemcomitans induces apoptosis of T lymphocytes by the Fas and Fas ligand pathway

MOLECULAR ORAL MICROBIOLOGY, Issue 5 2002
A. Nalbant
Actinobacillus actinomycetemcomitans expresses a number of toxins capable of inducing apoptotic cell death of T lymphocytes. However, the exact mechanism(s) has not been elucidated. The present study investigated the involvement of the Fas (CD95)-mediated apoptotic pathway in A. actinomycetemcomitans -induced T-cell apoptosis. To that end, peripheral blood mononuclear cells (PBMC) were cultured with or without A. actinomycetemcomitans cell-free culture supernatant (CFCS) for 0,96 h. The cells were then labeled with specific monoclonal antibodies and flow cytometry was performed. Results demonstrated up-regulation of Fas and activation of caspase-3 in T cells in response to A. actinomycetemcomitans CFCS. Monocytes were the only cells analyzed to express Fas ligand (FasL) constitutively, and this was further up-regulated in response to A. actinomycetemcomitans CFCS, while T cells expressed FasL only after this stimulation. Depletion of monocytes prior to stimulation with A. actinomycetemcomitans CFCS led to a marked decline in apoptosis. Blocking of Fas,FasL interactions with anti-Fas monoclonal antibody or Fas:Fc fusion protein lead to a significant decline, but not abolition, of T-cell apoptosis. Nearly all T cells expressed Bcl-2 at the outset of culture, and Bcl-2 expression declined in T cells stimulated with A. actinomycetemcomitans CFCS. Collectively, these data provide evidence for the induction of T-cell apoptosis by A. actinomycetemcomitans via the Fas-mediated pathway, involving caspase-3 and Bcl- 2. Moreover, this apoptotic response was dependent on the presence of monocytes. [source]

Monocytes from patients with myelodysplastic syndromes are more resistant to inhibition by thalidomide,

AMERICAN JOURNAL OF HEMATOLOGY, Issue 11 2009
Stef Meers
No abstract is available for this article. [source]

Functional immaturity of cord blood monocytes as detected by impaired response to hepatocyte growth factor

PEDIATRICS INTERNATIONAL, Issue 4 2001
Qi Jiang
AbstractBackground: Monocytes as antigen-presenting cells play an important role in host defense and transplantation. However, there are little reports on cord blood monocytes, and the role of monocytes in cord blood transplantation is largely unknown. Methods and Results: There are several cytokines affecting monocyte function. These include interferon-,, interleukin-4, interleukin-10, granulocyte macrophage-colony stimulating factor and hepatocyte growth factor (HGF). We investigated the effect of these cytokines on antigen-presenting capacity (APC) of cord and adult blood monocytes. Using either mononuclear cells or purified CD4+ T cells as responder cells, HGF enhanced APC of adult monocytes most effectively among these cytokines. In contrast, cord blood monocytes failed to respond to HGF. As HLA, costimulatory and adhesion molecules may affect APC function, we examined these antigens of monocytes following HGF stimulation. The HGF upregulated integrin ,5 subunit (CD49e) and intercellular adhesion molecule-1 (CD54) was expressed in adult blood monocytes, but not in cord blood. In kinetic studies, HGF downregulated c-met protein/HGF receptor expression of adult monocytes in lower concentrations and at shorter incubation time as compared with that of cord blood. Conclusions: The results suggest that impaired response of cord blood monocytes to HGF may be responsible, in large part, for their functional immaturity. [source]

ORIGINAL ARTICLE: Isolation of Non-Activated Monocytes from Human Umbilical Cord Blood

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 1 2010
Erik Normann
Problem, Methods for monocyte purification are common but few work with umbilical cord monocytes that do not activate the cell for subsequent culture analysis. Methods of study, The collection procedure avoids use of needles and procedures that variably activate blood clotting and uses a purification procedure that involves diluted Ficoll, autologous serum to remove platelets and 42% and 51% Percoll step gradients for the final purification. The resulting monocytes were stimulated with bacterial lipopolysaccharide and formalin-treated bacteria Escherichia coli and group B streptococci (GBS) to secrete TNF-, and IL-1,, measured by ELISA. Results, The purification procedure results in non-active but stimulation-competent monocytes with high yields (2.3,9 × 107 cells) and purity (from 70% to 98%). Conclusion, We describe a procedure that is easy, uses common reagents and provides a uniformly high yield and purity of non-activated fetal monocytes for studies of innate defense responses. [source]

Pregnancy-Specific Glycoproteins Function as Immunomodulators by Inducing Secretion of IL-10, IL-6 and TGF-,1 by Human Monocytes

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 4 2001
SARA K. SNYDER
PROBLEM: Low levels of pregnancy-specific glycoproteins (PSGs) in maternal serum have been correlated with complications of pregnancy. We investigated the ability of human PSGs to regulate in vitro production of cytokines. METHOD OF STUDY: Human monocytes and murine RAW 264.7 cells were treated with recombinant PSG1, PSG6, PSG11, or a truncated PSG6 consisting of only the N-terminal domain (PSG6N). Cytokine production in response to PSG-treatment was measured by ELISA and/or reverse transcriptase-PCR. RESULTS: All PSGs tested induced secretion of interleukin (IL)-10, IL-6 and transforming growth factor (TGF)-,1 by both human and murine cells, but not IL-1,, tumor necrosis factor (TNF)-, or IL-12. The N-terminal domain of PSG6 was sufficient for induction of monocyte cytokine secretion. Induction of IL-10 and IL-6 was preceded by an increase in the specific mRNAs. CONCLUSIONS: PSG1, PSG6, PSG6N, and PSG11 induce dose-dependent secretion of anti-inflammatory cytokines by human monocytes. Human and murine PSGs exhibit cross-species activity. Our results are consistent with a role for PSGs in modulation of the innate immune system. [source]

Peritubular Capillary Damage in Acute Humoral Rejection: An Ultrastructural Study on Human Renal Allografts

AMERICAN JOURNAL OF TRANSPLANTATION, Issue 12 2005
P. Lipták
The ultrastructural features of peritubular capillary (PC) damage was studied in 12 kidney allografts with acute humoral rejection (AHR). AHR manifested in diffuse linear PC staining for C4d, and histology consistent with Banff grade III in 7 recipients and Banff grade II in 5. Allografts with acute tubular necrosis served as controls. First biopsies (post-transplantation day 16.2 ± 2.2): The intra-capillary exudate comprised monocytes (59%), polymorphonuclears (14%), lymphocytes (12%) and not otherwise specified mononuclears (15%). Three patterns of focal PC endothelial injury were observed: lysis, an increased rate of apoptosis and fragmentation. No correlation was found between the respective damage types and the inflammatory cell types or the Banff grades. Controls revealed endothelial swelling, detachment from basement membrane and fragmentation. Follow-up biopsies: Monocytes transformed into macrophages intra-luminally. The reparative changes comprised endothelial cytoplasmic protrusions, binucleated endothelial cells and capillary sprouts. Early transplant capillaropathy and transplant glomerulopathy were noted in 2 recipients. Literature data indicate that lysis is mediated by anti-HLA alloantibodies; apoptosis, demonstrated first in the present study, may be induced by non-HLA-type anti-endothelial antibodies. Fragmentation is caused by ischemia. Ongoing endothelial injury leads to transplant capillaropathy and transplant glomerulopathy, the characteristic lesions of chronic rejection. [source]

Acute Cellular Rejection Predominated by Monocytes Is a Severe Form of Rejection in Human Renal Recipients With or Without Campath-1H (Alemtuzumab) Induction Therapy

AMERICAN JOURNAL OF TRANSPLANTATION, Issue 3 2005
Ping L. Zhang
Campath-1H has been used successfully for induction and has resulted in a low rate of acute cellular rejection (ACR) in renal transplantation in combination with various postoperative immunosuppression regimens. This study was undertaken to investigate the extent of monocyte involvement in ACR, with or without Campath-1H induction. We found that monocytes represented the majority of inflammatory cells in grades Ib or higher ACR, but not with Ia type of ACR, regardless of the status of Campath-1H induction. Cases of ACR, following Campath-1H induction, appear to demonstrate a ,pure form' of monocytic ACR, whereas monocytes were mixed with many other types of inflammatory cells in the cases of ACR in the absence of Campath-1H induction. In addition with Campath-1H induction, the cases of monocyte-predominant ACR were found to uniformly exhibit a good response to corticosteroid treatment. We conclude that monocyte-predominate ACR may represent a severe form of rejection, with or without Campath-1H treatment. [source]

The Guinea Fowl Spleen at Embryonic and Post-Hatch Periods

ANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 3 2006
B. I. Onyeanusi
Summary The spleen of the guinea fowl was bean-shaped but without a dented hilus. It is supplied by three short arteries that came from the ventral surface, two on the cranial end and one at the caudal end of the organ. The whole organ had a thin but tough capsule covering the outer surface except at the point of entry of the blood vessels. By day 18 of incubation, the spleen had a thin but well-defined capsule and internal to this been complete network of sinusoids filled with erythrocytes, lymphocytes and granulocytes. By day 19, dark and light staining zones, which could be termed red and white pulps, had appeared. By day 20, the granulocytes with a lot of granules within their cytoplasm, had become the biggest-sized cells in the spleen. At day 21, arteries and veins were noticed clearly in the spleen and many lymphocytes, few granulocytes and reticular cells surrounded these. Red pulp with its sinusoids was now distinct. A giant cell containing three nuclei was seen within the red pulp. At day 1 post-hatch, the capsule was at its greatest thickness so far and muscle cells were seen at the inner most part of the capsule. Granulocytes that had been a constant feature suddenly disappeared. At day 5, the small lymphocytes had dominated the large and medium-sized ones. By 2 weeks, the red and white pulps were virtually equal in distribution but by 3 weeks, the red pulp was convincingly greater. By 7 weeks, plasma cells had appeared in the peripheral splenic cords. Monocytes were observed in the sinusoids. Two germinal centres were identified for the first time in week 13 post-hatch. [source]

Differential expression of protease-activated receptors in monocytes from patients with primary antiphospholipid syndrome

ARTHRITIS & RHEUMATISM, Issue 3 2010
Chary López-Pedrera
Objective To investigate the expression of protease-activated receptors (PARs), their potential regulation by anticardiolipin antibodies (aCL), and their association with the expression of other molecules relevant to thrombosis in monocytes obtained from 62 patients with primary antiphospholipid syndrome (APS). Methods Monocytes were isolated from peripheral blood mononuclear cells by magnetic depletion of nonmonocytes. Expression of tissue factor (TF) and PARs 1,4 genes was measured by quantitative real-time reverse transcription,polymerase chain reaction. Cell surface TF and PARs 1,4 expression was analyzed by flow cytometry. For in vitro studies, purified normal monocytes were incubated with purified APS patient IgG, normal human serum IgG, or lipopolysaccharide, in the presence or absence of specific monoclonal antibodies anti,PAR-1 (ATAP2) or anti,PAR-2 (SAM11) to test the effect of blocking the active site of PAR-1 or PAR-2. Results Analysis of both mRNA and protein for the 4 PARs revealed significantly increased expression of PAR-2 as compared with the control groups. PAR-1 was significantly overexpressed in APS patients with thrombosis and in the control patients with thrombosis but without APS. PAR-3 expression was not significantly altered. PAR-4 expression was absent in all groups analyzed. In addition, we demonstrated a correlation between the levels of PAR-2 and the titers of IgG aCL, as well as parallel behavior of TF and PAR-2 expression. In vitro, IgG from APS patients significantly increased monocyte expression of PAR-1 and PAR-2. Inhibition studies suggested that there was direct cross-talk between TF and PAR-2, such that inhibition of PAR-2 prevented the aCL-induced expression of TF. Conclusion These results provide the first demonstration of increased expression of PARs in monocytes from patients with APS. Thus, PAR antagonists might have therapeutic potential as antithrombotic agents in APS. [source]