title: FluoroSpot Analysis of TLR-Activated Monocytes Reveals Several Distinct Cytokine-Secreting Subpopulations.
abstract: Monocytes have long been considered a heterogeneous group of cells both in terms of morphology and function. In humans, three distinct subsets have been described based on their differential expression of the cell surface markers CD14 and CD16. However, the relationship between these subsets and the production of cytokines has for the most part been based on ELISA measurements, making it difficult to draw conclusions as to their functional profile on the cellular level. In this study, we have investigated lipoteichoic acid (LTA)- and lipopolysaccharide (LPS)-induced cytokine secretion by monocytes using the FluoroSpot technique. This method measures the number of cytokine-secreting cells on the single-cell level and uses fluorescent detection, allowing for the simultaneous analysis of two cytokines from the same population of isolated cells. By this approach, human monocytes from healthy volunteers could be divided into several subgroups as IL-1β, IL-6, TNF-α and MIP-1β were secreted by larger populations of responding cells (25.9-39.2%) compared with the smaller populations of GM-CSF (9.1%), IL-10 (1.3%) and IL-12p40 (1.2%). Furthermore, when studying co-secretion in FluoroSpot, an intricate relationship between the monocytes secreting IL-1β and/or IL-6 and those secreting TNF-α, MIP-1β, GM-CSF, IL-10 and IL-12p40 was revealed. In this way, dissecting the secretion pattern of the monocytes in response to TLR-2 or TLR-4 stimulation, several subpopulations with distinct cytokine-secreting profiles could be identified.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Karolinska Institute, Stockholm, SwedenMabtech AB, Nacka Strand, SwedenDepartment of Clinical Science, Intervention and Technology, Karolinska Institute, Stockholm, Sweden.
authors.forename: C
authors.initials: C
authors.lastname: Smedman
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Karolinska Institute, Stockholm, SwedenMabtech AB, Nacka Strand, SwedenDepartment of Clinical Science, Intervention and Technology, Karolinska Institute, Stockholm, Sweden.
authors.forename: T
authors.initials: T
authors.lastname: Ernemar
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Karolinska Institute, Stockholm, SwedenMabtech AB, Nacka Strand, SwedenDepartment of Clinical Science, Intervention and Technology, Karolinska Institute, Stockholm, Sweden.
authors.forename: L
authors.initials: L
authors.lastname: Gudmundsdotter
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Karolinska Institute, Stockholm, SwedenMabtech AB, Nacka Strand, SwedenDepartment of Clinical Science, Intervention and Technology, Karolinska Institute, Stockholm, Sweden.
authors.forename: P
authors.initials: P
authors.lastname: Gille-Johnson
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Karolinska Institute, Stockholm, SwedenMabtech AB, Nacka Strand, SwedenDepartment of Clinical Science, Intervention and Technology, Karolinska Institute, Stockholm, Sweden.
authors.forename: A
authors.initials: A
authors.lastname: Somell
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Karolinska Institute, Stockholm, SwedenMabtech AB, Nacka Strand, SwedenDepartment of Clinical Science, Intervention and Technology, Karolinska Institute, Stockholm, Sweden.
authors.forename: K
authors.initials: K
authors.lastname: Nihlmark
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Karolinska Institute, Stockholm, SwedenMabtech AB, Nacka Strand, SwedenDepartment of Clinical Science, Intervention and Technology, Karolinska Institute, Stockholm, Sweden.
authors.forename: B
authors.initials: B
authors.lastname: Gårdlund
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Karolinska Institute, Stockholm, SwedenMabtech AB, Nacka Strand, SwedenDepartment of Clinical Science, Intervention and Technology, Karolinska Institute, Stockholm, Sweden.
authors.forename: J
authors.initials: J
authors.lastname: Andersson
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Karolinska Institute, Stockholm, SwedenMabtech AB, Nacka Strand, SwedenDepartment of Clinical Science, Intervention and Technology, Karolinska Institute, Stockholm, Sweden.
authors.forename: S
authors.initials: S
authors.lastname: Paulie
mesh_terms: D019070:Cell Lineage / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D054407:Chemokine CCL4 / Q000096:biosynthesis / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression / Q000276:immunology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D016753:Interleukin-10 / Q000096:biosynthesis / Q000378:metabolism; D053762:Interleukin-12 Subunit p40 / Q000096:biosynthesis / Q000378:metabolism; D053583:Interleukin-1beta / Q000096:biosynthesis / Q000378:metabolism; D015850:Interleukin-6 / Q000096:biosynthesis / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000145:classification / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D061251:Primary Cell Culture; D059010:Single-Cell Analysis / Q000379:methods*; D013682:Teichoic Acids / Q000494:pharmacology; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D054407:Chemokine CCL4; C508609:IL10 protein, human; C508600:IL6 protein, human; D053762:Interleukin-12 Subunit p40; D053583:Interleukin-1beta; D015850:Interleukin-6; D008070:Lipopolysaccharides; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D013682:Teichoic Acids; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; C009900:lipoteichoic acid; D016178:Granulocyte-Macrophage Colony-Stimulating Factor