Figure. 4. TNF production is markedly diminished in CD49b⁺ cells generated from Basoph8 x Tnf^fl/fl mice.

TNF (a and c) and IL-6 (b and d) concentrations in the supernatants of CD49b⁺ cells (1 × 10⁶ cells), following over-night sensitization with an IgE mAb to DNP (2 μg/ml) and 6 h of DNP-HSA (20 ng/ml) challenge (a, b) or TLR ligand stimulation (TLR1/2 [PAM 3CSK4, 250 ng/ml], TLR2 [HKLM, 1 × 10⁸/ml] or TLR4 [LPS, 1 μg/ml)]) (c, d). CD49b⁺ cells enriched from bone marrow-derived cells of Basoph8 x Tnf^fl/fl mice were markedly diminished compared with those observed after challenge of the control Tnf^fl/fl mice, whereas IL-6 production was unchanged. Data in a and b are shown as mean + SEM of the average of duplicate specimens with squares showing values obtained from individual experiments (n = 10 and n = 8 for cells from wild type mice in a and b, respectively, and n = 4 in a and b for cells from mice with basophil-derived TNF deficiency). Data in c and d are shown as mean + SEM of the average of duplicate specimens with squares showing values obtained from individual experiments. In c, n = 3 for medium, n = 7 for LPS and n = 5 for PAM 3CSK4 or HKLM for cells from wild type mice, and n = 3 for medium, n = 6 for LPS and n = 4 for PAM 3CSK4 or HKLM for cells from mice with basophil-derived TNF deficiency. In d, n = 5 medium, n = 6 for LPS and n = 5 for PAM 3CSK4 or HKLM for cells from wild type mice; and n = 7 for medium, n = 5 for LPS and n = 7 for PAM 3CSK4 or HKLM for cells from mice with basophil-derived TNF deficiency. P values by Mann-Whitney test. (e) Mean fluorescence intensity (MFI) for CD200R1 in bone marrow-derived basophils (FcεRIα⁺ CD49b⁺ c-Kit⁻) sensitized overnight with IgE mAb to DNP (2 μg/ml) and then challenged with DNP-HSA (20 ng/ml) for 1 h. CD200R1 expression was assessed by flow cytometry. The data in e were pooled from n = 3 independent experiments that were performed, with each condition tested in duplicate. Data are shown as means + SEM with squares showing values for cells obtained from individual mice (n = 4 for wild type cells and n = 8 for cells from mice with basophil-derived TNF deficiency). P values by Mann-Whitney test. (f, g) TNF production by peritoneal cell-derived mast cells (PCMCs) (f) and thyoglycollate-elicited peritoneal macrophages (g). PCMCs were sensitized overnight with IgE mAb to DNP (2 μg/ml) and then challenged with DNP-HSA (20 ng/ml) for 6 h. Peritoneal macrophages were stimulated with LPS (100 ng/ml) for 6 h. Data in f and g are shown as means + SEM with squares showing values from individual experiments (n = 13 and n = 12 for Tnf^fl/fl mice in f and g, respectively, and n = 4 for Basoph8 x Tnf^fl/fl for f and g) with each condition tested in duplicate. P values by Mann-Whitney test.