Figures & data
Figure 1. Adoptive transfer of alveolar macrophages. (A) Schematic of adoptive transfer protocol. AM from BAL of pregnant E14 and control mice were introduced by intra-nasal instillations to intact recipient female mice, followed by exposure of the recipients to TiO2 or vehicle the next day, and analysis by BAL 48 h afterwards. Thick red dotted line indicates intra-nasal TiO2 exposure; thin dotted lines indicate intra-nasal cell instillation. (B) Macrophages from pregnant mice confer the phenotype. Alveolar macrophages from BAL of pregnant E14 but not control mice confer increased responsiveness to TiO2 to intact recipient female mice. See schematic in for group assignments. Positive controls were pregnant mice with TiO2 exposure, as in prior studies. n = 6/group, three repeats. *p < 0.05 against any of the negative groups.
Figure 2. Pregnancy hormones inhibit particle uptake. (A) Detection of phagocytosing cells based on side-scatter. Left: Scattergram and histogram of vehicle control (PBS) sample. Right: Test sample with macrophages phagocytosing TiO2 particles for 2 h. Cells in charts were gated from general sample scatter to exclude debris. (B) Uptake of TiO2 particles in primary murine AM was reduced in cells from pregnant mice compared to controls. n = 12. *p < 0.01. Mean ± SEM. Representative experiment of three is shown. (C) Estradiol inhibits TiO2 particle uptake. Overnight pre-treatment of normal primary murine AM with 17β-estradiol (1 and 10 µg/ml) led to inhibition of particle uptake similar to that seen in cells from pregnant mice. Left (black bars): females, right (grey bars): males. Pooled average of two experiments; mean ± SEM. Effect significant at p < 0.05 for 10 µg/ml. (D) Dose-response inhibition of phagocytosis of TiO2 particles by RAW264.7 cells due to 17β-estradiol overnight pre-treatment. Average values of three representative repeats. Mean ± SEM. *p < 0.05, **p < 0.01. (E) Dose-response inhibition of phagocytosis of TiO2 particles in J774 cells by 17β-estradiol overnight pre-treatment. Average values from two representative repeats. Mean ± SEM. *p < 0.05. (F) Cell viability in estradiol-treated cultures determined by Annexin V and Cytox labeling to exclude both apoptotically- and necrotically-compromised cells. Representative sample from routine measures. Mean ± SEM.
Figure 3. Progesterone potentiates the effect of estradiol. While progesterone alone had minimal or no effect, it potentiated the action of estradiol. (A) RAW cells were pre-incubated with 0.1 or 1 µg/ml progesterone, with the positive control of 5 µg/ml estradiol +0.1 µg/ml progesterone, which was the only exposure that inhibited the uptake. (B) Cells were incubated with 5 µg/ml of estradiol with or without progesterone. (C) Average values from three experiments—comprising data in (A), (B), and data not shown elsewhere—using various combinations of estradiol (5 or 10 µg/ml) and progesterone (0.1 or 1 µg/ml). *p < 0.05 versus vehicle control. Mean ± SEM.
Figure A1. Microarray validation. Three samples per group of RAW cell RNA were tested for each of five genes to validate the results of the microarray analysis. Data are expressed as fold-change in 1 µg/ml estradiol-stimulated cells over control untreated cells (same for microarray and for PCR). All data are presented as mean ± SEM.
