scholarly journals In-VitroDifferentiation of Mature Dendritic Cells From Human Blood Monocytes

1998 ◽  
Vol 6 (1-2) ◽  
pp. 25-39 ◽  
Author(s):  
Robert Gieseler ◽  
Dirk Heise ◽  
Afsaneh Soruri ◽  
Peter Schwartz ◽  
J. Hinrich Peters
Keyword(s):  
Dendritic Cells ◽  
Human Blood ◽  
T Cell Response ◽  
Blood Monocytes ◽  
Gm Csf ◽  
Hla Dr ◽  
Hla Dq ◽  
Specific Stimulation ◽  
Antigen Presenting

Representing the most potent antigen-presenting cells, dendritic cells (DC) can now be generated from human blood monocytes. We recently presented a novel protocol employing GM-CSF, IL-4, and IFN-γto differentiate monocyte-derived DCin vitro. Here, such cells are characterized in detail. Cells in culture exhibited both dendritic and veiled morphologies, the former being adherent and the latter suspended. Phenotypically, they were CD1a-/dim, CD11a+, CD11b++, CD11c+, CD14dim/-, CD16a-/dim, CD18+, CD32dim/-, CD33+, CD40+, CD45R0+, CD50+, CD54+, CD64-/dim, CD68+, CD71+, CD80dim, CD86+/++, MHC class I++/+++HLA-DR++/+++HLA-DP+, and HLA-DQ+. The DC stimulated a strong allogeneic T-cell response, and further evidence for their autologous antigen-specific stimulation is discussed. Although resembling a mature CD 11c+CD45R0+blood DC subset identified earlier, their differentiation in the presence of the Thl and Th2 cytokines IFN-γand IL-4 indicates that these DC may conform to mature mucosal DC.

scholarly journals Impairments of Antigen-Presenting Cells in Pulmonary Tuberculosis

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Ludmila V. Sakhno ◽  
Ekaterina Ya. Shevela ◽  
Marina A. Tikhonova ◽  
Sergey D. Nikonov ◽  
Alexandr A. Ostanin ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Pulmonary Tuberculosis ◽  
Peripheral Blood ◽  
Antigen Presenting Cells ◽  
T Cell Response ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes ◽  
Gm Csf ◽  
Antigen Presenting

The phenotype and functional properties of antigen-presenting cells (APC), that is, circulating monocytes and generatedin vitromacrophages and dendritic cells, were investigated in the patients with pulmonary tuberculosis (TB) differing in lymphocyte reactivity toM. tuberculosisantigens (PPD-reactive versus PPD-anergic patients). We revealed the distinct impairments in patient APC functions. For example, the monocyte dysfunctions were displayed by low CD86 and HLA-DR expression, 2-fold increase in CD14+CD16+expression, the high numbers of IL-10-producing cells, and enhanced IL-10 and IL-6 production upon LPS-stimulation. The macrophages which werein vitrogenerated from peripheral blood monocytes under GM-CSF were characterized by Th1/Th2-balance shifting (downproduction of IFN-γcoupled with upproduction of IL-10) and by reducing of allostimulatory activity in mixed lymphocyte culture. The dendritic cells (generatedin vitrofrom peripheral blood monocytes upon GM-CSF + IFN-α) were characterized by impaired maturation/activation, a lower level of IFN-γproduction in conjunction with an enhanced capacity to produce IL-10 and IL-6, and a profound reduction of allostimulatory activity. The APC dysfunctions were found to be most prominent in PPD-anergic patients. The possible role of APC impairments in reducing the antigen-specific T-cell response toM. tuberculosiswas discussed.

Generation of dendritic cells using viral lysate of tumor cells in vitro.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15202-e15202
Author(s):  
Sergey A. Kolpakov ◽  
Elena P. Kolpakova ◽  
Anastasia O. Sitkovskaya ◽  
Elena Yu. Zlatnik ◽  
Svetlana Yu. Filippova ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Cytopathic Effect ◽  
Cytotoxic Lymphocytes ◽  
Blood Monocytes ◽  
U87mg Cell ◽  
Membrane Expression ◽  
Gm Csf ◽  
Highly Active ◽  
Hla Dr

e15202 Background: The purpose of the study was to investigate the effect of a new unclassified rotavirus on the effectiveness of maturation of dendritic cells (DC) and their activation of lymphocytes in vitro. Methods: Strain No.100 of the RVK virus was isolated by S.A. Kolpakov and characterized as rotavirus. U87MG cell lysate (glioblastoma) was obtained by incubation with 108 RVK particles in the DMEM medium with L-glutamine for 72 hours; a cytopathic effect was observed. Immature DCs were cultured for 7 days in the presence of IL-4 and GM-CSF. For antigen loading of DCs, we used the following options for 48 h: a) U87MG lysate obtained by repeated freeze-thaw cycles (TL); b) U87MG lysate obtained by co-cultivation with RVK (VTL); c) RVK. To assess the DC ability to activate autologous lymphocytes, they were co-cultured for 5 days in a 3:1 ratio. Results: The use of VTL culture for DC loading caused an increase in the expression of mature DC (mDC) markers compared to TL: the number of CD83+/CD86+ cells increased by more than 2 times, CD83+/CD80+ by 1.2 times, CD83+HLA-DR - by 5.5 times. With VTL, expression of markers of immature DCs (CD1a, CD14) was minimal. The use of RVK as an antigen induced the generation of DCs from monocytes, but their maturation was much less pronounced: a significant increase in the membrane expression of CD86, but not CD83, was determined. These DCs demonstrated a higher expression of markers of immature DCs, compared to stimulation with cell lysates (both TL and VTL): CD1a and CD14; CD80+/CD86+ level was the highest among all options. Analysis of the DC effect on co-cultivated lymphocytes showed that DCs loaded with RVK, both alone and as part of VTL, stimulated predominantly the NKT subpopulation. The same samples contained more T lymphocytes, activated CD4+ and CD8+ compared to samples stimulated by TL. However, the samples co-cultivated with VTL contained the maximal amount of CD4+/CD25+/CD127dim phenotypically corresponding to Tregs. Conclusions: The antigen loading of immature DCs with RVK alone causes their activation, but not maturation, which is not realized in typical terms of the DC generation from blood monocytes. The presence of RVK, including in VTL, has a stimulating effect on NKT lymphocytes suggesting the possible generation of specific highly active cytotoxic lymphocytes.

scholarly journals Cytokines in chronic inflammatory arthritis. IV. Granulocyte/macrophage colony-stimulating factor-mediated induction of class II MHC antigen on human monocytes: a possible role in rheumatoid arthritis.

1989 ◽  
Vol 170 (3) ◽  
pp. 865-875 ◽  
Author(s):  
J M Alvaro-Gracia ◽  
N J Zvaifler ◽  
G S Firestein
Keyword(s):  
Rheumatoid Arthritis ◽  
Class Ii ◽  
Tnf Alpha ◽  
Blood Monocytes ◽  
Ifn Gamma ◽  
Normal Peripheral Blood ◽  
Gm Csf ◽  
Hla Dr ◽  
Class Ii Mhc ◽  
Hla Dq

Granulocyte/macrophage CSF (GM-CSF) has recently been identified in rheumatoid arthritis (RA) synovial effusions. To study a potential role for GM-CSF and other cytokines on the induction of HLA-DR expression on monocytes and synovial macrophages, we analyzed the relative ability of recombinant human cytokines to induce the surface expression of class II MHC antigens on normal peripheral blood monocytes by FACS analysis. GM-CSF (800 U/ml) (mean fluorescence channel 2.54 +/- 0.33 times the control, p less than 0.001) and IFN-gamma (100 U/ml) (5.14 +/- 0.60, p less than 0.001) were the most potent inducers of HLA-DR. TNF-alpha and IL-4 also increased HLA-DR expression, although to a lesser degree [1.31 +/- 0.06 (p less than 0.02) and 1.20 +/- 0.03 (p less than 0.01), respectively]. IL-1 (40 U/ml), IL-2 (10 ng/ml), IL-3 (50 U/ml), IL-6 (100 U/ml), and CSF-1 (1,000 U/ml) did not affect surface HLA-DR density. GM-CSF also increased HLA-DR mRNA expression and surface HLA-DQ expression, but decreased CD14 (a monocyte/macrophage antigen) expression. The effect of GM-CSF on HLA-DR was not mediated by the generation of IFN-gamma in vitro because it was not blocked by anti-IFN-gamma mAb. GM-CSF was additive with IL-4 and low amounts (less than 3 U/ml) of IFN-gamma and synergistic with TNF-alpha. Because we have recently reported that supernatants of cultured RA synovial cells produce a non-IFN-gamma factor that induces HLA-DR on monocytes, we then attempted to neutralize this factor with specific anti-GM-CSF mAb. Four separate synovial tissue supernatants were studied, and the antibody neutralized the HLA-DR-inducing factor in each (p less than 0.01).

scholarly journals Leucine-Rich Repeat Kinase 2 Controls Inflammatory Cytokines Production through NF-κB Phosphorylation and Antigen Presentation in Bone Marrow-Derived Dendritic Cells

2020 ◽  
Vol 21 (5) ◽  
pp. 1890
Author(s):  
Makoto Kubo ◽  
Ryuichi Nagashima ◽  
Mitsue Kurihara ◽  
Fumitaka Kawakami ◽  
Tatsunori Maekawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Antigen Presentation ◽  
Inflammatory Cytokines ◽  
Immune Functions ◽  
Leucine Rich Repeat ◽  
Gm Csf ◽  
Macrophage Colony ◽  
Antigen Presenting

Leucine-rich repeat kinase 2 (LRRK2) is the causal molecule of familial Parkinson’s disease. Although the characteristics of LRRK2 have gradually been revealed, its true physiological functions remain unknown. LRRK2 is highly expressed in immune cells such as B2 cells and macrophages, suggesting that it plays important roles in the immune system. In the present study, we investigate the roles of LRRK2 in the immune functions of dendritic cells (DCs). Bone marrow-derived DCs from both C57BL/6 wild-type (WT) and LRRK2 knockout (KO) mice were induced by culture with granulocyte/macrophage-colony stimulating factor (GM/CSF) in vitro. We observed the differentiation of DCs, the phosphorylation of the transcriptional factors NF-κB, Erk1/2, and p-38 after lipopolysaccharide (LPS) stimulation and antigen-presenting ability by flow cytometry. We also analyzed the production of inflammatory cytokines by ELISA. During the observation period, there was no difference in DC differentiation between WT and LRRK2-KO mice. After LPS stimulation, phosphorylation of NF-κB was significantly increased in DCs from the KO mice. Large amounts of inflammatory cytokines were produced by DCs from KO mice after both stimulation with LPS and infection with Leishmania. CD4+ T-cells isolated from antigen-immunized mice proliferated to a significantly greater degree upon coculture with antigen-stimulated DCs from KO mice than upon coculture with DCs from WT mice. These results suggest that LRRK2 may play important roles in signal transduction and antigen presentation by DCs.

INTERDEPENDENCE BETWEEN THE PHENOTYPE OF DENDRITIC CELLS AND AMOUNTS OF BLOOD PROINFLAMMATORY MONOCYTES IN PATIENTS WITH KIDNEY CANCER

2019 ◽  
Vol 21 (4) ◽  
pp. 689-702
Author(s):  
A. A. Savchenko ◽  
A. G. Borisov ◽  
I. V. Kudryavtsev ◽  
A. V. Moshev
Keyword(s):  
Dendritic Cells ◽  
Kidney Cancer ◽  
Peripheral Blood ◽  
Receptor Expression ◽  
Autologous Serum ◽  
Autologous Tumor ◽  
Blood Monocytes ◽  
Inflammatory Monocytes ◽  
Antigen Presenting

The aim of the study was to investigate an interdependence between the phenotype of dendritic cells (DC) differentiated from monocytes and the number of pro-inflammatory monocytes in peripheral blood of patients with kidney cancer (KC). The study involved 28 patients at the age of 40-55 years suffering with KC (Т3N0М0, clear cell type) before surgical treatment. The diagnosis was verified histologically. 31 healthy agematched persons were examined as a control group. Mononuclear cells were isolated from heparinized venous blood by centrifugation in a Histopaque®-1077 density gradient followed by plastic adsorption in RPMI 1640 medium supplied with 10% autologous serum. Immature DCs (iDCs) were generated from blood monocytes by culturing for 5 days with GM-CSF and IFNα. Activation of DCs (mDCs) was induced by incubation with the tumor cell lysate and TNFα, followed by incubation for 48 hours. A tumor fragment was used to prepare the lysate of autologous tumor cells. Phenotyping of blood monocytes and DC at various maturation stages was performed by flow cytometry. The numbers of CD14+CD16+ monocytes in peripheral blood of KC patients were decreased (up to 42% of the total monocyte level) against the control ranges. In this regard, the analysis of the dependence between the phenotype of DCs differentiated from monocytes and the number of pro-inflammatory blood monocytes was carried out by comparing the groups with a high content of pro-inflammatory monocytes in the blood in KC patients (> 42%, near-control range) and low content (resp., < 42%). We have found that the contents of tolerogenic iDC in cell culture are increased in KC patients with low amounts of pro-inflammatory monocytes in blood (< 42%). A relatively increased expression of antigen-presenting and co-stimulatory molecules proved to be the specific feature of iDC phenotype in patients with high contents (> 42%) of proinflammatory monocytes in blood. The phenotype of dendritic cells in KC patients with different content of proinflammatory monocytes during maturation/activation showed more differences. In the patients with low levels of pro-inflammatory monocytes, the cell pool of in vitro maturing DCs was characterized by low level of CD86 and HLA-DR receptor expression, thus reflecting a weak co-stimulating and antigen-presenting activity. In the patients with high levels of pro-inflammatory monocytes in blood, the in vitro activated DCs showed higher level of functional activity using the above markers. The revealed differences in the DC phenotype and interrelations with amounts of blood monocyte subpopulations in KC patients may presume the programmed cell differentiation mechanisms depending on the microenvironment, under pathogenic conditions (i.e., in presence of malignant tumor growth).

scholarly journals Dendritic cells pulsed with protein antigens in vitro can prime antigen-specific, MHC-restricted T cells in situ.

1990 ◽  
Vol 172 (2) ◽  
pp. 631-640 ◽  
Author(s):  
K Inaba ◽  
J P Metlay ◽  
M T Crowley ◽  
R M Steinman
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell Response ◽  
Foreign Protein ◽  
Protein Antigens ◽  
Mhc Molecules ◽  
Antigen Presenting ◽  
Vacuolar System

T cells recognize peptides that are bound to MHC molecules on the surface of different types of antigen-presenting cells (APC). Antigen presentation most often is studied using T cells that have undergone priming in situ, or cell lines that have been chronically stimulated in vitro. The use of primed cells provides sufficient numbers of antigen-reactive lymphocytes for experimental study. A more complete understanding of immunogenicity, however, requires that one develop systems for studying the onset of a T cell response from unprimed lymphocytes, especially in situ. Here it is shown that mouse T cells can be reliably primed in situ using dendritic cells as APC. The dendritic cells were isolated from spleen, pulsed with protein antigens, and then administered to naive mice. Antigen-responsive T cells developed in the draining lymphoid tissue, and these T cells only recognized protein when presented on cells bearing the same MHC products as the original priming dendritic cells. In contrast, little or no priming was seen if antigen-pulsed spleen cells or peritoneal cells were injected. Since very small amounts of the foreign protein were visualized within endocytic vacuoles of antigen-pulsed dendritic cells, it is suggested that dendritic cells have a small but relevant vacuolar system for presenting antigens over a several day period in situ.

scholarly journals C-Reactive Protein Suppresses the Th17 Response Indirectly by Attenuating the Antigen Presentation Ability of Monocyte Derived Dendritic Cells in Experimental Autoimmune Encephalomyelitis

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhi-Yuan Shen ◽  
Yi Zheng ◽  
Maggie K. Pecsok ◽  
Ke Wang ◽  
Wei Li ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Blood Monocytes ◽  
C Reactive Protein ◽  
Autoimmune Encephalomyelitis ◽  
Th17 Response ◽  
Reactive Protein ◽  
Antigen Presenting ◽  
Experimental Autoimmune

Experimental autoimmune encephalomyelitis (EAE) is a classical murine model for Multiple Sclerosis (MS), a human autoimmune disease characterized by Th1 and Th17 responses. Numerous studies have reported that C-reactive protein (CRP) mitigates EAE severity, but studies on the relevant pathologic mechanisms are insufficient. Our previous study found that CRP suppresses Th1 response directly by receptor binding on naïve T cells; however, we did not observe the effect on Th17 response at that time; thus it remains unclear whether CRP could regulate Th17 response. In this study, we verified the downregulation of Th17 response by a single-dose CRP injection in MOG-immunized EAE mice in vivo while the direct and indirect effects of CRP on Th17 response were differentiated by comparing its actions on isolated CD4+ T cells and splenocytes in vitro, respectively. Moreover, the immune cell composition was examined in the blood and CNS (Central Nervous System), and a blood (monocytes) to CNS (dendritic cells) infiltration pathway is established in the course of EAE development. The infiltrated monocyte derived DCs (moDCs) were proved to be the only candidate antigen presenting cells to execute CRP’s function. Conversely, the decrease of Th17 responses caused by CRP disappeared in the above in vivo and in vitro studies with FcγR2B−/− mice, indicating that FcγR2B expressed on moDCs mediates CRP function. Furthermore, peripheral blood monocytes were isolated and induced to establish moDCs, which were used to demonstrate that the antigen presenting ability of moDCs was attenuated by CRP through FcγR2B, and then NF-κB and ERK signaling pathways were manifested to be involved in this regulation. Ultimately, we perfected and enriched the mechanism studies of CRP in EAE remission, so we are more convinced that CRP plays a key role in protecting against EAE development, which may be a potential therapeutic target for the treatment of MS in human.

scholarly journals Azithromycin drives in vitro GM-CSF/IL-4-induced differentiation of human blood monocytes toward dendritic-like cells with regulatory properties

2011 ◽  
Vol 91 (2) ◽  
pp. 229-243 ◽  
Author(s):  
Darija Stupin Polančec ◽  
Vesna Munić Kos ◽  
Mihailo Banjanac ◽  
Mila Vrančić ◽  
Snježana Čužić ◽  
...  
Keyword(s):  
Human Blood ◽  
Blood Monocytes ◽  
Induced Differentiation ◽  
Gm Csf ◽  
Regulatory Properties

Serial Analysis of Gene Expression in Human Monocyte-Derived Dendritic Cells

Blood ◽  
1999 ◽  
Vol 94 (3) ◽  
pp. 845-852 ◽  
Author(s):  
Shin-ichi Hashimoto ◽  
Takuji Suzuki ◽  
Hong-Yan Dong ◽  
Shigenori Nagai ◽  
Nobuyuki Yamazaki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dendritic Cells ◽  
Cell Structure ◽  
Human Monocyte ◽  
Interleukin 4 ◽  
Maturation Stage ◽  
Blood Monocytes ◽  
Gm Csf ◽  
Highly Expressed Genes

Dendritic cells (DCs) are professional antigen-presenting cells in the immune system and can be generated in vitro from hematopoietic progenitor cells in the bone marrow, CD34+ cord blood cells, precursor cells in the peripheral blood, and blood monocytes by culturing with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4, and tumor necrosis factor-. We have performed serial analysis of gene expression (SAGE) in DCs derived from human blood monocytes. A total of 58,540 tag sequences from a DC complementary DNA (cDNA) library represented more than 17,000 different genes, and these data were compared with SAGE analysis of tags from monocytes (Mo) and GM-CSF–induced macrophages (M◊). Many of the genes that were differentially expressed in DCs were identified as genes encoding proteins related to cell structure and cell motility. Interestingly, the highly expressed genes in DCs encode chemokines such as TARC, MDC, and MCP-4, which preferentially chemoattract Th2-type lymphocytes. Although DCs have been considered to be very heterogeneous, the identification of specific genes expressed in human Mo-derived DCs should provide candidate genes to define subsets of, the function of, and the maturation stage of DCs and possibly also to diagnose diseases in which DCs play a significant role, such as autoimmune diseases and neoplasms. This study represents the first extensive gene expression analysis in any type of DCs.

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