Article Figures & Data
Figures
- 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.
- 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.
- 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.
- 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.
