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Induction of Dendritic Cell-like Phenotype in Macrophages during Foam Cell Formation-GSE7138

Purpose

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Hypothesis

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Experimental Design

Human blood was drawn from the antecubital veins of healthy blood donors and provided as buffy coats by the Virginia Blood Services (Richmond, VA). The mononuclear fractions were pooled from four unidentified donors to decrease individual variations in monocytes. Mixed peripheral blood mononuclear cells (PBMCs) were isolated by Histopaque 1.077 (Sigma Diagnostics, Inc., St. Louis, MO). Following centrifugation, the mononuclear layer was removed and washed with PBS containing 0.02% ethylenediaminetetraacetate (EDTA). The pellet was resuspended in 1X H-lyse Buffer (R&D Systems Inc., Minneapolis, MN), and washed with wash buffer. PBMCs contain mainly monocytes and lymphocytes as well as platelets that tend to be associated with blood monocytes. From these PBMCs, monocytes were isolated using a negative selection monocyte isolation kit and LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the isolated fraction was > 97% as estimated by flow cytometry using anti-CD14 (data not shown). Although these cells are often called “untouched” monocytes and thought to show little activation, the gene chip analysis conducted on these cells shows massive changes in gene expression compared to PBMCs (see below).Monocytes were cultured in Macrophage Serum-Free Medium (MSFM, Invitro-gen, Carlsbad, CA) in the presence of 1% media supplement nutridoma-HU (Roche Molecular Biochemicals, Indianapolis, IN) and 100 nM M-CSF for 6 days, after which the cells showed the expected morphological signs of macrophage differentiation. These macrophages were incubated either with 100 nM CCL2 or CXCL1 for 5 hours. CXCL1 was selected because we have previously found that it is an important arrest chemokine for monocytes in vitro and in atherosclerotic arteries in vivo. CCL2 was chosen because mice lacking CCL2 or its receptor CCR2 are relatively resistant to atherosclerosis, suggesting a role in macrophage recruitment, differentiation and/or survival. Human monocyte-derived macrophages (MDM) were also incubated with native LDL, oxidized LDL (oxLDL) or minimally modified LDL (mmLDL) (each at a concentration of 100 ug/ml) for 2 days to induce foam cell formation. Foam cell formation was verified by oil red O staining (figure 1) and by determining their cholesterol and cholesterol ester content. OxLDL and mmLDL were prepared from the same native LDL for each experiment as described. Control experiments were conducted on macrophages cultured in M-CSF without LDL for an additional 2 days. Two separate sets of monocytes were incubated with CXCL4 (100 nM) for 6 days, another procedure known to induce macrophage differentiation (29), with and without oxLDL to induce foam cell formation. RNA was extracted from cells in all 11 conditions (table 1) and gene expression was measured in duplicates at the University of Virginia Gene Expression Core Facility using Affymetrix equipment.

Experimental Variables

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Controls

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Methods

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Additional Information
  • Cho HJ, Shashkin P, Gleissner CA, Dunson D et al. Induction of dendritic cell-like phenotype in macrophages during foam cell formation. Physiol Genomics2007 Apr 24;29(2):149-60. PMID: 17244792
  • Microarray
    Affymetrix HG-U133A
    22 Samples Loaded: 22
    Human (Homo sapiens)
    PBMC , Monocytes , Macrophages
    Healthy
    Sample Set Spreadsheet
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    Samples Preview
    Sample ID !Sample_title healthy blood donors Culture conditions
    GSM170908 PBMC #1 True PBMC
    GSM170910 PBMC #2 True PBMC
    GSM170913 Monocytes #1 True Monocyte
    GSM170915 Monocytes #2 True Monocyte
    GSM170916 Macrophages 6d #1 True Macrophage D6
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    File
    sampleset4000052_sampleannotations.csv
    Sample Set Spreadsheet
    Raw Signal
    Raw Signal
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    All fields are editable except the "Sample ID" column. To edit a cell, click within the cell. To edit a "date" cell, click on the calendar icon. To cancel an edit, press the ESC key.

    Sample ID !Sample Title Healthy blood donors Culture conditions
    GSM170908
    PBMC #1
    True
    PBMC
    GSM170910
    PBMC #2
    True
    PBMC
    GSM170913
    Monocytes #1
    True
    Monocyte
    GSM170915
    Monocytes #2
    True
    Monocyte
    GSM170916
    Macrophages 6d #1
    True
    Macrophage D6
    GSM170918
    Macrophages 6d #2
    True
    Macrophage D6
    GSM170920
    CCL2 #1
    True
    CCL2
    GSM170922
    CCL2 #2
    True
    CCL2
    GSM170932
    CXCL1 #1
    True
    CXCL1
    GSM170933
    CXCL1 #2
    True
    CXCL1
    GSM170935
    Macrophages 8d #1
    True
    Macrophage D8
    GSM170936
    Macrophages 8d #2
    True
    Macrophage D8
    GSM170937
    LDL #1
    True
    LDL
    GSM170938
    LDL #2
    True
    LDL
    GSM170941
    mmLDL #1
    True
    mmLDL
    GSM170942
    mmLDL #2
    True
    mmLDL
    GSM170943
    oxLDL #1
    True
    oxLDL
    GSM170944
    oxLDL #2
    True
    oxLDL
    GSM170969
    CXCL4 #1
    True
    CXCL4
    GSM170970
    CXCL4 #2
    True
    CXCL4
    GSM170971
    CXCL4+oxLDL #1
    True
    CXCL4+oxLDL
    GSM170972
    CXCL4+oxLDL #2
    True
    CXCL4+oxLDL

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