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Blood, Vol. 112, Issue 9, 3762-3771, November 1, 2008
Human basophils activated by mast cell–derived IL-3 express retinaldehyde dehydrogenase-II and produce the immunoregulatory mediator retinoic acid
Blood Spiegl et al.
112: 3762
Supplemental materials for: Spiegl et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 58.9 KB)
- Table S1. Mass spectroscopy analysis of peptide fragments of the 55 kDa proteins of activated basophils (PDF, 46.7 KB)
- Table S2. Fold change of RALDH2 mRNA levels in stimulated basophils (PDF, 479 KB)
- Figure S1. Time and dose dependent induction of functional RALDH2 (JPG, 58.6 KB)
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(A) Time course of induction of RALDH2 and RA-producing capacity. Upper panel: Basophils were cultured for indicated time periods with IL-3 (50 ng/ml); retinal (10 nM) was added for the last 2 hours of culture. Collected supernatants were used in a 1 to 2 dilution in RA-reporter assays. Mean values (± s.d.) of triplicates of a representative experiment are shown. Lower panel: Kinetics of RALDH2 induction. Western blot analysis of RALDH2 expression in basophils cultured for 0–72 h with IL-3. (B) Dose-dependent induction of RALDH2 and RA synthesis by IL-3. Upper panel: Basophils were incubated for 24 h with different concentration of IL-3 and co-cultured with reporter cells in the absence or presence of retinol (30 nM). Retinol and medium alone are shown as controls (no cells). Lower panel: Dose-dependent induction of RALDH2 by IL-3. Basophils were stimulated with different concentrations of IL-3 for 24 h. RALDH2 expression was analysed by western blotting using anti-RALDH2 antibody.
- Figure S2. The capacity to express RALDH2 is restricted to basophils among different primary human myeloid and lymphoid cells (JPG, 82.6 KB)
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RALDH2 expression in distinct leukocyte subsets as assessed by Western blotting. Primary cells were isolated from peripheral blood and cultured as follows. (A) Myeloid dendritic cells (mDCs) were stimulated for 24 h without (buffer) or with LPS (100 ng/ml). Monocytes were freshly isolated (f.i.), non-stimulated (buffer) or cultured for 48 h with the stimuli indicated: IFN-γ (1000 U/ml); M-CSF, GM-CSF, and IL-3 (50 ng/ml); IL-4 and IL-13 (20 ng/ml). (B) Freshly isolated naïve and memory CD4+ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies (1 µg/ml) for the time periods indicated. Invariant NKT cells (iNKT) were stimulated for 24 h with OCH (100 ng/ml), αGC (100 ng/ml) or PHA (1 µg/ml). (C) Strongly polarized Th1 and Th2 clones were stimulated with plate-bound anti-CD3 and anti-CD28 (1 µg/ml) for the time periods indicated. (D) Purified B cells were cultured for 0-5 days without stimulation. (E) Expression of full length and truncated isoforms of RALDH2 in different cell lines. Basophils (Ba), non-stimulated or stimulated with IL-3 (± IL-3), are included as controls. Actin is shown as loading control.
- Figure S3. Paracrine action of basophil-derived RA on naïve T-helper cells (JPG, 98.3 KB)
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Purified naïve CD4+ T cells cultured without (T cells alone) or co-cultured with basophils in a 1:1 ratio (Co-culture) were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies (1 µg/ml) in the absence or presence of the agents indicated (10 ng/ml IL-3, 100 nM AGN, 10 nM RA) for 10 days. T cells were stained for (A) β7-integrin, (B) CD38, or (C) CCR9 and analysed by flow cytometry. Molt-4 cells were stained for CCR9 as positive control. Bar charts are given in delta mean fluorescence intensities (ΔMFI). Mean values (± s.d.) of triplicates of a representative experiment are shown on the left. Examples of histograms are shown on the right.
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