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Blood, Vol. 113, Issue 12, 2816-2825, March 19, 2009
Basophil effector function and homeostasis during helminth infection
Blood Ohnmacht and Voehringer
113: 2816
Supplemental materials for: Ohnmacht and Voehringer
Files in this Data Supplement:
- Figure S7. IgE-induced release of IL-5 from basophils (JPG, 12.3 KB)
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Bone marrow derived basophils were sensitized with anti-TNP specific IgE (white bar) or not (black bar) and cultured for 16h in the presence of TNP-BSA to induce IL-5 protein release. IL-5 in the supernatant was measured with a commercial ELISA kit (OptEIA, Becton Dickinson). The bars show the mean+s.d. from triplicates. P<0.0001 by Student’s t-test.
- Figure S8. Eosinophils and basophils are the main IL-4 expressing cells in the lung of 4get/rag−∕− mice (JPG, 43.1 KB)
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4get/rag−∕− mice were infected with N. brasiliensis and either depleted of basophils by anti-Thy1.2 treatment (depleted) or not (control). Single cell suspensions of total lung tissue were analyzed on day 4 after infection by flow cytometry after staining for Siglec-F (to detect eosinophils) and IgE (to detect basophils). The gate in the dot plots on the left indicates the total population of IL-4/eGFP expressing cells which consist mainly of eosinophils (Siglec-F+IgE−) and basophils (Siglec-F-IgE+) as indicated by the dot plots on the right.
- Figure S9. Differentiation of alternatively activated marophages is IL-4/IL-13 dependent (JPG, 11.4 KB)
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Wild-type or IL-4/IL-13–deficient mice on BALB/c background were infected with N. brasiliensis and RT-PCR analysis for Fizz1, Ym1, and hprt was performed on day 9 after infection with cDNA from total lung tissue. PCR was performed with 3-fold serially diluted cDNA templates as described in Materials and Methods.
- Figure S10. Flow cytometric analysis of basophil depletion in reconstituted 4get/rag−∕− mice (JPG, 25.3 KB)
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4get/rag−∕− Thy1.2 mice were reconstituted with 7 × 107 lymphocytes from IL-4/IL-13–deficient Thy1.1 mice and depleted of basophils by anti-Thy1.2 administration. Dot plots show the frequency of basophils (IgE+CD4− cells) in spleen and lung of anti–Thy1.2-treated and control mice.
- Figure S11. Retroviral expression of IL-3 from T cells promotes mast cell and basophil differentiation (JPG, 26.6 KB)
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CD4 T cells were stimulated with anti-TCR antibody and transfected with retroviral constructs encoding either IL-3-IRES-GFP (RV-IL-3) or IRES-GFP alone (RV-control). Culture supernatants were collected two days after transfection and added at 1:100 dilution to bone marrow cultures. As positive control cultures were setup with 0.3 ng/ml recombinant IL-3. 11 days later cultures were analyzed by flow cytometry. The dot plots demonstrate that supernatants from RV-IL-3 transfected cells contained biologically active IL-3 which promotes differentiation of mast cells (c-Kit+FcεRI+) and basophils (c-Kit−FceRI+).
- Figure S12. Expression of IL-4/eGFP and various surface markers on dendritic cells (JPG, 35.3 KB)
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Splenocytes from 4get mice were stained with anti-CD11c and anti-Thy1.2, anti-FcεRI or anti–c-Kit. Dot plots show that DCs (CD11c+ cells) are negative for IL-4/eGFP, Thy1.2, and FcεRI. However, they express low levels of c-Kit.
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