Differential Expression Of CD14 Research Articles

Blood monocyte profiles in COPD patients with PiMM and PiZZ α1-antitrypsin.

Human blood monocytes are divided into populations based on the differential expression of CD14 and CD16 receptors: CD14 + CD16(classical), CD14 + CD16 + (intermediate), and CD14-CD16+ (non-classical). Given their functional differences and their role in pathogenesis of chronic obstructive pulmonary disease (COPD), monocyte profiling is of clinical interest. Here we investigated blood monocyte subsets in clinically stable COPD patients with alpha1-antitrypsin (AAT) deficiency (PiZZ, n=7) and with normal AAT variant (PiMM, n=7). Peripheral whole blood was collected in sodium heparin tubes and incubated with LPS (from E. coli; 1μg/ml) or placebo for 6hat 37°C, 5% CO2. To profile monocyte subsets we performed flow cytometry analysis based on HLA-DR and CD14/CD16 staining. HLA-DR+subsets of cells did not differ between PiZZ and PiMM COPD, and healthy controls (n=7), used as a reference. Monocyte profiling, which express the CD14 and CD16, but not the HLA-DR (HLA-DR-) showed that intermediate monocytes subset was lowest in PiZZ group, and almost totally disappeared from blood treated with LPS. The non-classical subset was almost absent in PiZZ patients independently of LPS treatment. Recent studies demonstrate that non-classical monocytes exhibit a unique ability to protect the vascular endothelium under both homeostatic and inflammatory conditions whereas intermediate monocytes are recruited at a later stage of inflammation, and are associated with secretion of cytokines/chemokines and wound healing. Evident alterations in blood monocyte subsets together with a partial reduction of AAT levels, an important anti-inflammatory protein, can be key factors for the early manifestation of emphysema in some PiZZ AATD carriers.

Precise Delineation and Transcriptional Characterization of Bovine Blood Dendritic-Cell and Monocyte Subsets.

A clear-cut delineation of bovine bona fide dendritic cells (DC) from monocytes has proved challenging, given the high phenotypic and functional plasticity of these innate immune cells and the marked phenotypic differences between species. Here, we demonstrate that, based on expression of Flt3, CD172a, CD13, and CD4, a precise identification of bovine blood conventional DC type 1 and 2 (cDC1, cDC2), plasmacytoid DC (pDC), and monocytes is possible with cDC1 being Flt3+CD172adimCD13+CD4−, cDC2 being Flt3+CD172a+CD13−CD4−, pDC being Flt3+CD172adimCD13−CD4+, and monocytes being Flt3−CD172ahighCD13−CD4−. The phenotype of these subsets was characterized in further detail, and a subset-specific differential expression of CD2, CD5, CD11b, CD11c, CD14, CD16, CD26, CD62L, CD71, CD163, and CD205 was found. Subset identity was confirmed by transcriptomic analysis and subset-specific transcription of conserved key genes. We also sorted monocyte subsets based on their differential expression of CD14 and CD16. Classical monocytes (CD14+CD16−) clustered clearly apart from the two CD16+ monocyte subsets probably representing intermediate and non-classical monocytes described in human. The transcriptomic data also revealed differential gene transcription for molecules involved in antigen presentation, pathogen sensing, and migration, and therefore gives insights into functional differences between bovine DC and monocyte subsets. The identification of cell-type- and subset-specific gene transcription will assist in the quest for “marker molecules” that—when targeted by flow cytometry—will greatly facilitate research on bovine DC and monocytes. Overall, species comparisons will elucidate basic principles of DC and monocyte biology and will help to translate experimental findings from one species to another.

Monocytic myeloid-derived suppressor cells generated from rhesus macaque bone marrow enrich for regulatory T cells

Putative monocytic myeloid-derived suppressor cells (mMDSC; lineage−HLA-DR−/lo) were generated in 7-day cultures from normal rhesus macaque bone marrow (BM) cells in GM-CSF and IL-6. Three subsets were identified based on their differential expression of CD14, CD33, CD34 and CD11b. Following flow sorting, assessment of the capacity of these subsets to suppress anti-CD3/CD28-stimulated CD4 and CD8 T cell proliferation revealed that the most potent population was CD14hiCD33−/loCD34loCD11bhi. These BM-derived mMDSC markedly increased the incidence of CD4+CD25+CD127−Foxp3+ regulatory T cells in responder T cell populations. They offer potential value in testing the therapeutic efficacy of immunoregulatory mMDSC for the promotion of tolerance in nonhuman primate transplant models.

A Single-Cell Gene-Expression Profile Reveals Inter-Cellular Heterogeneity within Human Monocyte Subsets.

Human monocytes are a heterogeneous cell population classified into three different subsets: Classical CD14++CD16-, intermediate CD14++CD16+, and non-classical CD14+CD16++ monocytes. These subsets are distinguished by their differential expression of CD14 and CD16, and unique gene expression profile. So far, the variation in inter-cellular gene expression within the monocyte subsets is largely unknown. In this study, the cellular variation within each human monocyte subset from a single healthy donor was described by using a novel single-cell PCR gene-expression analysis tool. We investigated 86 different genes mainly encoding cell surface markers, and proteins involved in immune regulation. Within the three human monocyte subsets, our descriptive findings show multimodal expression of key immune response genes, such as CD40, NFⱪB1, RELA, TLR4, TLR8 and TLR9. Furthermore, we discovered one subgroup of cells within the classical monocytes, which showed alterations of 22 genes e.g. IRF8, CD40, CSF1R, NFⱪB1, RELA and TNF. Additionally one subgroup within the intermediate and non-classical monocytes also displayed distinct gene signatures by altered expression of 8 and 6 genes, respectively. Hence the three monocyte subsets can be further subdivided according to activation status and differentiation, independently of the traditional classification based on cell surface markers. Demonstrating the use and the ability to discover cell heterogeneity within defined populations of human monocytes is of great importance, and can be useful in unravelling inter-cellular variation in leukocyte populations, identifying subpopulations involved in disease pathogenesis and help tailor new therapies.

A1.48 Enhanced expression of CD11c on non-classical CD16+ peripheral blood monocytes in early rheumatoid arthritis

Background and ObjectivesClassical (CM), non-classical (NCM) and intermediate populations of human monocytes are characterised by differential expression of CD14 and CD16. NCM and IM are potent antigen presenting cells and...

Monocyte Mayhem

Atherosclerosis is a lipoprotein disorder with inflammatory, vascular, and hemodynamic determinants. Total white blood cell count and subpopulations including granulocytes, monocytes, T cells, and bone marrow-derived precursors have been implicated in atherosclerotic cardiovascular disease (CVD).1,2 Although these associations may inform biological processes, they have not, to date, permitted specific mechanistic understanding of subsets of leukocytes involved nor have they advanced risk prediction and therapeutic targeting or monitoring in the clinic. Although short-lived in circulation, blood monocytes are proposed to play a specific role in atherosclerosis because (1) blood monocytes are the major source of tissue macrophages; (2) monocyte-derived macrophages and dendritic cells are the predominant leukocytes in atherosclerotic lesion; and (3) macrophages/dendritic cells have a variety of important proinflammatory (inflammatory recruitment/activation of other leukocytes and vascular cells, foam cell formation and lipid accumulation, fibrous cap erosion, and plaque rupture) and anti-inflammatory (phagocytosis, efferocytosis, and leukocyte egress) actions in experimental atherosclerotic lesions.1,3,4 Despite these mechanistic insights from human pathology and experimental studies in rodents, there have been disparate findings in studies of total monocyte counts and CVD.5,6 Article see p 122 There are several challenges to our understanding of the role of monocytes in atherosclerosis.7,8 First, circulating monocytes are heterogeneous due to subtype differentiation in bone marrow. The regulation and rate of subpopulation recruitment as well as their roles in atherosclerosis are uncertain. Second, monocyte subpopulations are typically defined by differential expression of specific cell surface antigens including receptors that modulate their function, for example, chemokine receptors chemokine (C-C motif) receptor 2 (CCR2), CX3C chemokine receptor 1 (CX3CR1), and C-C chemokine receptor type 5 (CCR5), as well as adhesion molecule integrin receptors very late antigen-4 (also called integrin α4β1) and LFA-1(integrin αLβ …

SuperSAGE evidence for CD14++CD16+ monocytes as a third monocyte subset

AbstractMonocytes are a heterogeneous cell population with subset-specific functions and phenotypes. The differential expression of CD14 and CD16 distinguishes classical CD14++CD16−, intermediate CD14++CD16+, and nonclassical CD14+CD16++ monocytes. Current knowledge on human monocyte heterogeneity is still incomplete: while it is increasingly acknowledged that CD14++CD16+ monocytes are of outstanding significance in 2 global health issues, namely HIV-1 infection and atherosclerosis, CD14++CD16+ monocytes remain the most poorly characterized subset so far. We therefore developed a method to purify the 3 monocyte subsets from human blood and analyzed their transcriptomes using SuperSAGE in combination with high-throughput sequencing. Analysis of 5 487 603 tags revealed unique identifiers of CD14++CD16+ monocytes, delineating these cells from the 2 other monocyte subsets. Gene Ontology (GO) enrichment analysis suggests diverse immunologic functions, linking CD14++CD16+ monocytes to Ag processing and presentation (eg, CD74, HLA-DR, IFI30, CTSB), to inflammation and monocyte activation (eg, TGFB1, AIF1, PTPN6), and to angiogenesis (eg, TIE2, CD105). In conclusion, we provide genetic evidence for a distinct role of CD14++CD16+ monocytes in human immunity. After CD14++CD16+ monocytes have earlier been discussed as a potential therapeutic target in inflammatory diseases, we are hopeful that our data will spur further research in the field of monocyte heterogeneity.

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

CD14, a key candidate gene associated with a specific immune response to cockroach

Sensitization to cockroach allergen is one of the strongest predictors of asthma morbidity, especially among African Americans. Our aims were to determine the genomic basis of cockroach sensitization and the specific response to cockroach antigen. We investigated the Th1/Th2 cytokine profile of co-cultured plasmacytoid dendritic cells (pDCs) and CD4+ T cells and the 'transcript signature' of the immune response to cockroach antigen using high-throughput expression profiling of co-cultured cells. We observed significantly elevated levels of IL-13, IL-10, and TNF-alpha, but undetectable levels of IL-12p70 and IFN-alpha, when cultures were exposed to crude cockroach antigen. A significant difference was observed for IL-13 between cockroach-allergic and non-allergic individuals (P=0.039). Microarray analyses demonstrated a greater response at 48 h compared with 4 h, with 50 genes being uniquely expressed in cockroach antigen-treated cells, including CD14, S100A8, CCL8, and IFI44L. The increased CD14 expression was further observed in purified pDCs, human monocytic THP-1 cells, and the supernatant of co-cultured pDCs and CD4+ T cells on exposure to cockroach extract. Furthermore, the most differential expression of CD14 between cockroach allergy and non-cockroach allergy was only observed among individuals with the CC 'high-risk' genotype of the CD14-260C/T. Ingenuity Pathways Analysis analyses suggested the IFN signalling as the most significant canonical pathway. Our results suggest that these differentially expressed genes, particularly CD14, and genes in the IFN signalling pathway may be important candidates for further investigation of their role in the immune response to cockroach allergen.

Heterogeneity of Human Macrophages in Culture and in Atherosclerotic Plaques

Research suggests that monocytes differentiate into unique lineage-determined macrophage subpopulations in response to the local cytokine environment. The present study evaluated the atherogenic potential of two divergent lineage-determined human monocyte-derived macrophage subpopulations. Monocytes were differentiated for 7 days in the presence of alternative macrophage development cytokines: granulocyte-macrophage colony-stimulating factor to produce granulocyte-macrophage-CSF macrophages (GM-Mac), or macrophage colony-stimulating factor (M-CSF) to produce M-Mac. Gene chip analyses of three monocyte donors demonstrated differential expression of inflammatory and cholesterol homeostasis genes in the macrophage subpopulations. Quantitative PCR confirmed a fivefold elevation in the expression of genes that promote reverse cholesterol transport (PPAR-gamma, LXR-alpha, and ABCG1) and macrophage emigration from lesions (CCR7) in GM-Mac compared to that in M-Mac. Immunocytochemistry confirmed enhanced expression of the proinflammatory marker CD14 in M-Mac relative to GM-Mac. M-Mac spontaneously accumulated cholesterol when incubated with unmodified low-density lipoprotein whereas GM-Mac only accumulated similar levels of cholesterol after protein kinase C activation. Immunostained human coronary arteries showed that macrophages with similar antigen expression to that of M-Mac (CD68(+)/CD14(+)) were predominant within atherosclerotic lesions whereas macrophages with antigen expression similar to GM-Mac (CD68(+)/CD14(-)) were predominant in areas devoid of disease. The identification of macrophage subpopulations with different gene expression patterns and, thus, different potentials for promoting atherosclerosis has important experimental and clinical implications and could prove to be a valuable finding in developing therapeutic interventions in diseases dependent on macrophage function.

Ex vivo characterization of human thymic dendritic cell subsets

Interactions between thymic dendritic cells (DC) and thymocytes are critical for proper development of T-cells. We identified human thymic DC populations on the basis of CD123, CD11c and CD14 expression. High levels of CD123 (IL-3R) and CD45RA defined the plasmacytoid DC (pDC) subset. Human thymic CD11c + DC expressed CD45RO and myeloid-related markers (CD13, CD33 and CD11b). CD11c + DC could be separated into two main subsets based on differential expression of CD14: CD11c + CD14 − and CD11c + CD14 + cells. Spontaneous production of IL-10 and IFN γ without exogenous stimulation, was observed in the three DC subsets. Important phenotype modifications were observed in pDC cultures supplemented with IL-3. A down-regulation of CD123 and appearance of myeloid markers such as CD11b and CD11c on CD45RA + cells was noticed within the first 48 h; at a later time there was a shift from CD45RA to CD45RO expression, as well as appearance of CD14 expression. CD11c + cells emerging in pDC culture did not express high levels of HLA-DR, CD83 and co-stimulatory molecules. This suggests an in vitro evolution of human thymic pDC toward a myeloid phenotype found in the CD11c + subset of thymic DC.

Large variations in human foam cell formation in individuals: a fully autologous in vitro assay based on the quantitative analysis of cellular neutral lipids

The transformation of monocyte-derived macrophages into lipid-laden foam cells constitutes a characteristic and crucial event in the development of the earliest atherosclerotic lesions. We investigated whether the propensity to form foam cells varies among individuals. We developed a fully autologous foam cell assay based on a recently developed novel culture technique for human monocyte-derived macrophages (Wintergerst ES, Jelk J, Asmis, R. Differential expression of CD14, CD36 and the LDL receptor on human monocyte-derived macrophages. A novel cell culture system to study macrophage differentiation and heterogeneity, Histochem. Cell Biol. 1998;110:231–241). Thin layer chromatography and laser densitometry were used to determine cholesterol, triglyceride and cholesteryl ester levels in human macrophages. Aggregated LDL obtained by vortexing was found to be a reproducible stimulus of foam cell formation in human macrophages. In our hands, Cu 2+-oxidized LDL also induced cholesteryl ester accumulation, but only when vortexed. We found that foam cell formation in an individual varied by less than 25% over a 10-month period. In contrast, we observed a sevenfold difference in foam cell formation among eight male volunteers. The transfer of foam cells into culture medium with freshly thawed autologous serum resulted in a 75% regression within 1 week, independent of the amount of cellular cholesteryl esters accumulated. Foam cell formation correlated neither to serum nor to cellular cholesterol and triglyceride levels. The propensity to form foam cells could therefore represent a novel indicator of individual risk of atherogenesis.

Differential expression of CD14, CD36 and the LDL receptor on human monocyte-derived macrophages. A novel cell culture system to study macrophage differentiation and heterogeneity.

Macrophages are key players in many aspects of human physiology and disease. It has been hypothesized that in a given microenvironment monocytes differentiate into specific subpopulations with distinct functions. In order to study the role of macrophage heterogeneity in atherogenesis, we established a novel isolation and culture technique for human monocyte-derived macrophages. The present technique does not select for monocyte subpopulations prior to the onset of differentiation. Monocytes were cultured for 2 weeks in the presence of autologous lymphocytes before being plated quantitatively. They differentiated into mature macrophages in terms of morphology, lipid composition, and biological activity. Based on phagocytic activity as well as on the expression of CD14, CD36, and the low-density lipoprotein (LDL) receptor, we have identified macrophage subpopulations that may play distinct roles in atherogenesis. While virtually all adherence-purified monocytes expressed CD14, CD36, and the LDL-R, we characterized three subpopulations of macrophages based on the expression of these antigens: CD36+CD14-LDL-R-(58+/-12%), CD36+CD14+LDL-R+(18+/-5%), the remaining cells being CD36-CD14- LDL-R-. The first two subsets decreased in size during further differentiation (51+/-12% and 8+/-3%, respectively). Our culture technique may also serve as a good model for studying the implications of macrophage heterogeneity in diseases other than atherosclerosis.