Figure 9.

Co-localization of DC and CD4⁺ lymphocytes within bronchovascular infiltrates coincides with increased mRNA expression of IL-12 and IFN-γ in CCR2^+/+ mice. (A) CCR2^+/+ mice were inoculated IT with C. neoformans and lungs removed at day 10 post-infection. Samples were snap-frozen and later stained for anti-MHC Class II (I-A^d; black alkaline phosphatase reaction) to identify DC with characteristic morphology and anti-CD4 (red peroxidase reaction) to identify CD4⁺ lymphocytes. Photomicrograph (×200 magnification) depicting a bronchovascular infiltrate adjacent to an airway (AW). Note the extensive co-localization of DC with CD4⁺ lymphocytes. At higher magnification (see inset, ×1000), individual DC are identified interacting with multiple CD4⁺ lymphocytes. (B–D) RNA was prepared from lung homogenates obtained from CCR2^+/+ mice and CCR2^−/− mice at baseline and day 14 post-IT infection with C. neoformans. A separate RNA preparation from uninfected CCR2^+/+ mice (n=5) was used as a reference standard. RT-PCR was performed to evaluate the expression of (B) IL-12p35, (C) IFN-γ and (D) IL-4. CCR2^+/+mice, black bar; CCR2^−/− mice, gray bar. Data represents a mean ± SEM of 5 mice assayed individually per time-point in 2 experiments; * p<0.05 by unpaired student t-test vs. Day 0 (uninfected) mice of the same CCR2 expression profile, ‡p<0.05 by unpaired student t-test when values from CCR2^+/+ mice vs. CCR2^−/− mice were compared at the designated time point.