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Fungal and Parasitic Infections

Involvement of CD14 and Toll-Like Receptors in Activation of Human Monocytes by Aspergillus fumigatus Hyphae

J. E. Wang, A. Warris, E. A. Ellingsen, P. F. Jørgensen, T. H. Flo, T. Espevik, R. Solberg, P. E. Verweij, A. O. Aasen
J. E. Wang
Institute for Surgical Research, Rikshospitalet—National Hospital, N-0027 Oslo,
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A. Warris
Department of Medical Microbiology, University Medical Center, Nijmegen, The Netherlands
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E. A. Ellingsen
Institute for Surgical Research, Rikshospitalet—National Hospital, N-0027 Oslo,
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P. F. Jørgensen
Institute for Surgical Research, Rikshospitalet—National Hospital, N-0027 Oslo,
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T. H. Flo
Institute of Cancer Research and Molecular Biology, Norwegian University of Science and Technology, Trondheim, Norway, and
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T. Espevik
Institute of Cancer Research and Molecular Biology, Norwegian University of Science and Technology, Trondheim, Norway, and
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R. Solberg
Department of Pharmacology, School of Pharmacy, University of Oslo, N-0316 Oslo, and
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P. E. Verweij
Department of Medical Microbiology, University Medical Center, Nijmegen, The Netherlands
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A. O. Aasen
Institute for Surgical Research, Rikshospitalet—National Hospital, N-0027 Oslo,
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DOI: 10.1128/IAI.69.4.2402-2406.2001
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    Fig. 1.

    Expression of mRNAs for TNF-α, IL-1β, IL-6, and β-actin in various leukocyte populations 6 h after incubation of whole blood in the absence (lanes 1 to 4) or presence of hyphal fragments isolated from A. fumigatus (lanes 5 to 8) orS. prolificans (lanes 9 to 12). Monocytes (CD14+), granulocytes (CD15+), T cells (CD2+), and B cells (CD19+) were isolated by immunomagnetic separation. mRNA from these cells was isolated by oligo(dT)25-coated magnetic beads, reverse transcribed, and analyzed for transcripts encoding TNF-α (A [443 bp]), IL-1β (B [802 bp]), IL-6 (C [628 bp]), and β-actin (D [660 bp]) by PCR. Unstim., unstimulated.

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    Fig. 2.

    Dependency on plasma for stimulation of TNF-α release by human monocytes (four donors) by hyphal fragments from A. fumigatus. (A) Human monocytes isolated from whole blood were spiked with autologous plasma (0, 1, 5, or 10%) and cultured for 8 h in the absence or presence of hyphal fragments. (B) Monocytes were stimulated with hyphal fragments in the presence of 5% untreated plasma or 5% plasma that had been heat treated at 56°C for 30 min. Supernatants were subsequently analyzed for TNF-α by EIA. Data are means ± standard errors of four experiments. ∗, significantly lower (P < 0.05) than values obtained with untreated plasma, as calculated by Student's t test.

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    Fig. 3.

    Influence of blockade of CD14, TLR4, and TLR2 on primary adherent monocytes by MAbs on stimulation of TNF-α production byAspergillus hyphae (A) and LPS (B). Human monocytes were pretreated with different concentrations (1, 3, or 10 μg/ml) of 18D11 (anti-CD14), HTA125 (anti-TLR4), TL2.1 (anti-TLR2), or an IgG1 control antibody for 30 min at room temperature under serum-free conditions. Hyphal fragments and 5% plasma (or LPS [10 ng/ml]) were subsequently added, followed by incubation for 8 h. Supernatants were analyzed for TNF-α by EIA. Results are means ± standard errors of six to nine experiments. ∗, significantly lower (P < 0.05) than values obtained with control antibodies, as calculated by analysis of variance.

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    Fig. 4.

    Influence of a synthetic analog of Rhodobacter capsulatus lipid A (B975) on TNF-α release from primary adherent human monocytes induced by Aspergillus hyphae or LPS (10 ng/ml). Monocytes were pretreated with various doses of B975 (0, 0.1, 1, 10, or 100 nM) for 30 min prior to addition ofAspergillus hyphae or LPS (10 ng/ml). After 8 h of stimulation, supernatants were isolated and analyzed for TNF-α by EIA. Results are means ± standard deviations of triplicate samples in one typical experiment performed three times. ∗, significantly lower (P < 0.05) than values obtained in the absence of B975, as calculated by analysis of variance.

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Involvement of CD14 and Toll-Like Receptors in Activation of Human Monocytes by Aspergillus fumigatus Hyphae
J. E. Wang, A. Warris, E. A. Ellingsen, P. F. Jørgensen, T. H. Flo, T. Espevik, R. Solberg, P. E. Verweij, A. O. Aasen
Infection and Immunity Apr 2001, 69 (4) 2402-2406; DOI: 10.1128/IAI.69.4.2402-2406.2001

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Involvement of CD14 and Toll-Like Receptors in Activation of Human Monocytes by Aspergillus fumigatus Hyphae
J. E. Wang, A. Warris, E. A. Ellingsen, P. F. Jørgensen, T. H. Flo, T. Espevik, R. Solberg, P. E. Verweij, A. O. Aasen
Infection and Immunity Apr 2001, 69 (4) 2402-2406; DOI: 10.1128/IAI.69.4.2402-2406.2001
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KEYWORDS

Aspergillus fumigatus
Drosophila Proteins
Lipopolysaccharide Receptors
Membrane Glycoproteins
monocytes
Receptors, Cell Surface

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