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First published online 22 June 2004
doi: 10.1242/jcs.01162

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IL-4 alone without the involvement of GM-CSF transforms human peripheral blood monocytes to a CD1a^dim, CD83⁺ myeloid dendritic cell subset

Keshab Chandra Roy^*, Gautam Bandyopadhyay, Srabanti Rakshit, Mitali Ray and Santu Bandyopadhyay

The Division of Immunology, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Kolkata, 700 032, India

View larger version (88K): [in a new window]   Fig. 1. Endogenous GM-CSF is undetectable in IL-4-DC. (A) Detection of intracellular GM-CSF in monocytes (freshly isolated or cultured for 1 day and 7 days in the presence of IL-4) before and after stimulation with LPS. Relevant cells were surface stained for the expression of CD11c (FL2), permeabilized, stained for intracellular GM-CSF (FL1). Quadrants were set after staining with isotype-matched control mAbs. The proportion of positive cells are given in each quadrant. One representative experiment of three is shown. (B) Specificity and sensitivity of intracellular GM-CSF staining was demonstrated by surface staining fresh monocytes (stimulated with medium or LPS) with CD11c followed by staining for GM-CSF without permeabilization. (C) Detection of GM-CSF transcripts by RT-PCR. Total RNA was extracted from relevant cells, 500 ng (i) and 2000 ng (ii) RNA were used to analyse the expression of mRNA for GM-CSF and ß-actin. Data are representative of three similar experiments. (D) Yields of immature and mature cells. The numbers of monocytes plated on day 0 was set equal to 100%. Percentages indicate the numbers of recovered cells on day 7 for immature and on day 9 of culture for mature cells. Data shown are from three separate experiments; error bars indicate s.d. (E) Phase contrast microscopy of TNF--stimulated (48 hours) mature cells. Note the numerous veiled processes in IL-4-DC and GM-IL-4-DC. Magnification x300.

View larger version (42K): [in a new window]   Fig. 2. Phenotypic characterization of Med-MO, IL-4-DC and GM-IL-4-DC. (A) Maturation was induced by stimulation with TNF-a (20 ng/ml) for 2 days. Large cells were gated. The fluorescence of gated cells is depicted in the histograms. Dotted lines in each panel represent staining with isotype-matched irrelevant control Igs. Each row of histograms represents FACS profiles of one individual side-by-side comparative experiments. The differentially expressed molecule is CD1a. The expression of CD1a is barely detectable in IL-4-DC compared with GM-IL-4-DC. Each marker was probed in at least three separate experiments. (B) Neutralization of GM-CSF by anti-GM-CSF mAb inhibits CD1a expression of GM-IL-4-DC. Freshly purified monocytes containing >90% CD14+ cells (top panel; dotted line represents staining with isotype-matched control Ig, unbroken line represents staining with anti-CD14 mAb) were stained for surface expression of CD1a and GM-CSF receptor chain (CD116) immediately and after culture in media alone, or media containing GM-CSF (800 U/ml) plus IL-4 (1000 U/ml) and graded concentrations of neutralizing anti GM-CSF mAb (1.0 to 10.0 µg/ml), or media containing IL-4 alone. Data shown are from three separate representative experiments.

View larger version (50K): [in a new window]   Fig. 3. Detection of myeloid DC cell surface-associated transcripts in IL-4-DC by RT-PCR. Cells were induced to mature for 48 hours with TNF- (20 ng/ml). Total RNA was extracted and analysed for expression of mRNA for CD1a, CD1b, CD1c, CD83, HLA-DR, CCR7, CXCR4 and ß-actin. Data shown are representative of four similar experiments.

View larger version (33K): [in a new window]   Fig. 4. Comparative cytofluorographic analysis of phagocytic activity, and intracellular myeloperoxidase (MPO) of freshly isolated and cultured monocytes. Cells were incubated with 3 µM FITC-latex beads for 30 minutes at 37°C (A) or stained with anti-MPO mAb after permeabilization (B) and examined by flow cytometry. Dotted lines in the histograms of MPO represent staining with isotype-matched irrelevant control Ig. Data shown are representative of three separate experiments.

View larger version (68K): [in a new window]   Fig. 5. Detection of cytokine transcripts in IL-4-DC by RT-PCR. Cells were left unstimulated or stimulated with LPS (1 µg/ml) for 6 hours, total RNA were extracted and were analysed for expression of mRNA for IL-12, IL-10, IFN-, TNF-, and ß-actin. Data are representative of three similar experiments.

View larger version (44K): [in a new window]   Fig. 6. Intracellular cytokine/chemokine analysis of IL-4-DC by flow cytometry. (A) Cells were activated with LPS (1 µg/ml) for 18 hours. Brefeldin A was added to the cultures for the last 4 hours and then intracellular IFN-/IL-4 (upper row) and MlP-1/IL-8 (lower row) were analysed. The percentages of the respective cytokine-producing cells are shown in each dot plot profile. Quadrants were set according to the fluorescence intensities of FITC- and PE-conjugated isotype-matched control Igs with irrelevant specificity. (B) Cells were left untreated or stimulated with anti-CD40 mAb (10 µg/ml) or LPS (1.0 µg/ml) plus IFN- (100 ng/ml) for 18 hours to detect intracellular IL-12/IL-13. Quadrants were set after staining with isotype matched control mAbs. The percent positive cells are given in each quadrant. The flow cytometry results are representative of three similar experiments.

View larger version (21K): [in a new window]   Fig. 7. Immunostimulatory functions of immature, and mature, IL-4-DC and GM-IL-4-DC. MLR was performed with both immature (A) and mature (B,C) stimulator cells as shown. Maturation of stimulator cells was induced either by TNF- (B) or CD40 ligation (C). Proliferation was measured by [3H]thymidine incorporation (cpm). The background proliferation of T cells alone was always less than 350 cpm. The data represent mean ± s.d. of triplicate wells. One representative experiment of three is shown.

View larger version (36K): [in a new window]   Fig. 8. Mature IL-4-DC and GM-IL-4-DC induce Th1 cytokines during primary allogeneic MLR. Mature IL-4-DCs and GM-IL-4-DCs (maturation was induced by TNF-) were co-cultured with allogeneic NPBMC depleted of HLA-DR+ cells, NK cells and B cells as described in Materials and Methods at 1:10 ratio for 6 days, activated with 2.4 µg/ml PHA plus 1.0 ng/ml PMA for 6 hours. Brefeldin A was added for the last 4 hours. Cells were washed, permeabilized and stained for detection of intracellular cytokines using FITC- or PE-conjugated specific antibodies. Lymphocytes were gated and intracellular cytokines were analysed by flow cytometry. Numbers within histograms represent percentages within marker bounds. Markers were set employing isotype control staining. One of four experiments with similar results is shown.

View larger version (40K): [in a new window]   Fig. 9. Immature IL-4-DC and GM-IL-4-DC induce IL-10 but suppress IL-2 and IFN- from allogeneic bulk T cells after repeated stimulations. IL-4-DCs and GM-IL-4-DCs were co-cultured with allogeneic NPBMC depleted of HLA-DR+ cells, NK cells and B cells for 6 days at a ratio of 1:10. Stimulation with APCs was repeated weekly from the same donor under the same conditions for three times as described in Materials and Methods. Six days after the last stimulation with APCs, cells were washed, re-stimulated with 2.4 µg/ml PHA plus 1.0 ng/ml PMA for 6 hours. Brefeldin A was added for the last 4 hours. Cells were permeabilized, stained for intracellular cytokines using FITC- or PE-conjugated specific mAbs. Lymphocytes were gated and intracellular cytokines were analysed by flow cytometry. Numbers within histograms represent percentages within marker bounds. Markers were set employing isotype control staining. One of three experiments is shown.

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