Figure 3
The CD11b+Gr1int cells do not migrate to the lung-draining lymph nodes (LNs) and lipopolysaccharide (LPS) instillation in the lung does not inhibit CD4+ T-cell proliferation in the LNs. (a) The lin− population in bone marrow cells was enriched using a lineage depletion kit and phenotypic analysis was performed by flow cytometry. In addition, shown is the phenotypic analysis of the lin+ fraction. The lin− population of bone marrow cells was enriched from enhanced green fluorescent protein (EGFP) transgenic mice and transferred intravenously (i.v.) into naive mice. Mice were divided into two groups and one group then received three daily treatments of 10 μg LPS, whereas the other group was left untreated. The top right panel shows the presence of GFP+ cells in the lung after transfer of GFP+ lin− cells. The forward vs. side light scatter pattern revealed a distinct non-lymphocytic population of cells in which CD11b- and Gr1-expressing cells were concentrated (data not shown). This population of cells was gated on for subsequent analyses. Within the GFP gate, the percentage of cells expressing intermediate (int) levels of Gr1 was determined in the presence or absence of LPS (lower panel). (b) CD11c+ and CD11b+ cells were purified from the lung cells obtained from LPS-treated mice. Expression of CCR7 on low autofluorescent CD11c+ and CD11b+ cells was detected by flow cytometry. (c) Expression of CD11b and Gr1 on LN cells measured by flow cytometry with or without LPS treatment of mice. The histogram shows CCR7 levels on CD11c+ cells obtained from LN cells of LPS-treated mice. Thin gray lines indicate staining with appropriate isotype control antibody and the overlay represents expression of the CCR7 molecules. The last panel shows a ∼30-fold increase in the number of CD11c+Gr1− cells in the LNs in LPS-treated mice. Data represent average values obtained from three independent experiments±s.d. **P<0.005. (d) T cell receptor (TCR) transgenic CD4+ T cells were isolated from the spleens of DO11.10 mice. The cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and adoptively transferred to either naive or LPS-treated mice (four daily treatments with 10 μg per treatment of LPS). Beginning 1 day after adoptive transfer, the mice received ovalbumin/cholera toxin (OVA/CT) once per day for 2 days. After 24 h, the number of KJ-126+ (DO11.10), CFSE-expressing cells in the lung-draining LNs was determined (upper panel), and the degree of cell proliferation was quantitated based upon CFSE dilution (lower panels). Results shown are representative of two independent experiments.
