Figure 3

Lipopolysaccharide (LPS) enhances the migration of lung dendritic cells (DCs) to the mediastinal lymph nodes (MLNs) and promotes T helper 2 (Th2) differentiation. (a) CD4⁺ T cells from Thy1.1^+/+ RAG-1^−/− WT15 T-cell receptor (TCR) transgenic mice were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and injected (2 × 10⁶ per mouse) into postasthmatic mice. Mice were exposed 12 h later to LPS or phosphate-buffered saline (PBS), and MLN cells were analyzed by fluorescence-activated cell sorting (FACS) 3 days later following staining with antibodies to Thy1.1 and CD4. Data show representative FACS profiles after gating on Thy1.1⁺ WT15 cells. The numbers indicate the frequency (%) of Thy1.1⁺ CD4⁺ T cells that have undergone ⩾5 divisions (left panels). Data are expressed as mean±s.d. and are representative of two independent experiments. Data show the fold increase in the number of WT15 cells recovered in the MLNs of LPS-exposed mice when compared with those recovered in PBS-exposed animals (right panel). Results are expressed as mean±s.d.; n=10 mice per group pooled from two experiments. (b) CD4⁺ T cells from WT15 and DO.11.10 TCR transgenic mice were labeled with CFSE and co-injected (2 × 10⁶ cells of each per mouse) into postasthmatic mice. Mice were exposed 12 h later to either LPS or PBS alone or to both LPS and ovalbumin (OVA). MLN cells were analyzed by FACS 3 days later following staining with antibodies to Thy1.1, KJ.1-26 and CD4. Data show representative FACS profiles after gating on KJ.1-26⁺ and Thy1.1⁺ CD4⁺ cells of two independent experiments with n=5 mice per group. The gates corresponding to WT15 and DO.11.10 T cells are indicated. (c–e) Postasthmatic mice were exposed to LPS or PBS together with fluorescent latex beads 21 days after LACK (Leishmania analog of the receptors of activated C kinase) challenge. The MLN cells were analyzed by FACS 3 days later. (c) Data show representative FACS profiles after staining with antibody to CD11c. The numbers indicate cell frequency within the indicated gate and are expressed as mean±s.e.m.; n=5 mice per group. Two independent experiments were performed. (d, e) Bead⁺ DCs were sorted and 10⁴ cells were incubated with 10⁵ CFSE-labeled Thy1.1^+/+ RAG-1^−/− WT15 CD4⁺ T cells for 7 days. (d) Data show representative FACS profiles after gating on Thy1.1⁺ CD4⁺ T cells. The numbers indicate the frequency of cells that have undergone ⩾6 divisions. (e) Cellular supernatants were assessed for interleukin (IL)-13, IL-5 and interferon-γ (IFN-γ) content by multiplex analysis using Cytometric Bead Array (CBA). Data are expressed as mean±s.d. of triplicate wells from one representative experiment out of two. *P<0.05. (f, g) Postasthmatic langerin-GFP mice were exposed to LPS or PBS together with fluorescent latex beads and MLN cells were analyzed 3 days later. (f) Data show representative FACS profiles after staining with antibodies to CD11c and CD11b. The numbers indicate the mean frequency±s.d. of cells in the indicated gates. (g) Data show the mean number±s.e.m. of langerin⁻ (dark bars) and langerin⁺ (empty bars) bead⁺ DCs in PBS- and LPS-exposed mice (left panel). Data show the MFI±s.e.m. upon staining with antibodies to CD80 and CD86 after gating on langerin⁻ (dark bars) or langerin⁺ (empty bars) bead⁺ DCs (right panels). n=10 mice per group pooled from two experiments. *P<0.05; **P<0.01; NS, not significant.

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