Immunostimulatory potential of β-lactoglobulin preparations

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Presentation transcript:

Immunostimulatory potential of β-lactoglobulin preparations Susanne Brix, MS, Lionel Bovetto, PhD, Rodolphe Fritsché, PhD, Vibeke Barkholt, PhD, Hanne Frøkiaer, PhD Journal of Allergy and Clinical Immunology Volume 112, Issue 6, Pages 1216-1222 (December 2003) DOI: 10.1016/j.jaci.2003.08.047

FIG 1 Induction of proliferation and intracellular thiol increase in cow's-milk–naive immune cells after in vitro exposure to β-LG. Splenocytes and MLNs from BALB/c mice were cultured with various concentrations of the different milk proteins and OVA. A, Proliferation of spleen cells.B, Proliferation of MLNs.C, Intracellular thiol level in splenocytes cultured with 1 mg/mL protein. Data are mean ± SD for proliferation, n = 6. Differences in cell proliferation toward the proteins were analyzed for significance using 1-way ANOVA. ∗∗∗P < .001 by Tukey's multiple-comparisons test. Journal of Allergy and Clinical Immunology 2003 112, 1216-1222DOI: (10.1016/j.jaci.2003.08.047)

FIG 2 Purification of β-LG from raw milk using non-denaturating conditions. A, Anionic ion-exchange chromatography of whey proteins. B, Size-exclusion chromatography (Superdex 75 pg column) of the β-LG variant A+B fraction from A. C, SDS-PAGE (4%-12% NuPAGE) after silver staining: molecular weight marker (lane 1), unfractionated whey proteins (lane 2), the purity of the β-LG preparation before (lane 3), and after size-exclusion chromatography (lane 4). The protein amount applied to the SDS-PAGE was ∼2.5 μg. Journal of Allergy and Clinical Immunology 2003 112, 1216-1222DOI: (10.1016/j.jaci.2003.08.047)

FIG 3 No immunomodulatory effects of β-LG from raw milk. Splenocytes and MLNs from cow's-milk–naive BALB/c mice were cultured with an optimal dose of 1 mg/mL protein. A, Proliferation of splenocytes and MLNs. Data (mean ± SD, n = 6) are stimulation index calculated as counts per minute (cpm) of antigen-stimulated cultures divided by that of unstimulated cultures. The cpm of unstimulated cultures was 332 ± 88 for spleen cells and 342 ± 38 for MLNs. B, Cytokine production by spleen cells (mean ± SD, n = 6). C, Increase in cellular thiol in spleen cells. All data are representative of 2 independent experiments. Differences in cell proliferation and cytokine production between proteins were tested using 1-way ANOVA. ∗∗P < .01. ∗∗∗P < .001 by Tukey's multiple-comparisons test. S, Sigma-Aldrich. Journal of Allergy and Clinical Immunology 2003 112, 1216-1222DOI: (10.1016/j.jaci.2003.08.047)

FIG 4 Size-exclusion chromatography of Sigma β-LG separates the immunostimulatory component from β-LG. The Sigma β-LG preparation (20 mg/mL) was fractionated on a Superdex 75 pg column using 150 mmol/L ammonium acetate, pH 6.0. The absorbance was measured at 280 nm (solid black line). The insert presents the whole chromatogram. Proliferation in response to each collected fraction and the unfractionated β-LG was tested on MLNs from cow's-milk–naive BALB/c mice. The proliferation data (mean, n = 6) are shown as stimulation index (black bars). Results are representative of 2 independent experiments. Journal of Allergy and Clinical Immunology 2003 112, 1216-1222DOI: (10.1016/j.jaci.2003.08.047)

FIG 5 The immunostimulatory component of the Sigma β-LG preparation induces cytokine production in murine DC cultures. MBM cells were in vitro expanded by GM-CSF for 8 days and then cultured with 1 mg/mL antigen or 1 μg/mL LPS (E coli O26:B6). After 19 hours, the supernatants were harvested, and the cytokines TNF-α, IL-6, IL-1β, and IL-10 were measured by ELISA. Data (mean ± SD) are derived from 1 experiment using cells from 2 mice and tested in duplicate wells. The F6 and F15 were obtained from size-exclusion chromatography of the Sigma β-LG preparation (Fig 4). S: Sigma-Aldrich; AF: Arla Foods, Denmark. Journal of Allergy and Clinical Immunology 2003 112, 1216-1222DOI: (10.1016/j.jaci.2003.08.047)

FIG 6 The cellular response to Sigma β-LG is reduced in the LPS low-responder strain C3H/HeJ. Proliferation of MLNs from C3H/HeJ and C3H/HeOuJ mice (n = 6) cultured with 1 mg/mL antigen or 10 μg/mL LPS (E coli O55:B5). The F6 and F15 were obtained from size-exclusion chromatography of the Sigma β-LG preparation (Fig 4). ∗∗∗P < .001 by 2-way ANOVA followed by the Bonferroni multiple-comparisons test. NS: Not significant; S: Sigma-Aldrich; AF: Arla Foods, Denmark. Journal of Allergy and Clinical Immunology 2003 112, 1216-1222DOI: (10.1016/j.jaci.2003.08.047)