Characterization of Human Lung Airway APC Subsets

The respiratory system is a complex network of many cell types, including subsets of macrophages and dendritic cels, that work together to maintain steady-state respiration. Due to limitations in acquiring cells from healthy human lung, these subsets remain poorly characterized transcriptionally and phenotypically. We set out to systemically identify these subsets in human airways, by developing a schema of isolating large numbers of cells by whole lung bronchoalveolar lavage. Six subsets of phagocytic antigen presenting cells were consistently observed, which varied in their ability to internalize bacterial particles. Subsets could be further separated by their inherent capacities to upregulate CD83, CD86, and CCR7. Whole genome transcriptional profiling revealed a clade of “true dendritic cells” distinct from a “macrophage/monocyte” clade. Each clade, and each member of both clades, could be discerned by specific genes of increased expression, which would serve as markers for future studies in healthy and diseased states. 6 antigen presenting cell subsets were sorted from 7 different human lung donors. In addition, immature monocyte-derived dendritic cells and macrophages were generated from monocytes of 6 human blood donors. Monocyte-derived dendritic cells were generated by exposure to IL-4 and GM-CSF, while monocyte-derived macrophages were generated by exposure to GM-CSF alone. Immature macrophages and dendritic cells were sorted as the lung cells were. A fraction of the monocyte-derived cells from the same blood donors were further matured with LPS exposure prior to sorting. Samples were processed into purified total RNA, which was reverse transcribed into cDNA and further amplified. Reverse transcription and amplification occurred in two batches of samples. Positive and negative controls (composed of blood PBMC) for RT and amplification were included with both batches, but were not used in analysis. Samples were fragmented and biotinylated for hybridization with Human genome U133 Plus 2.0 arrays. Array data was log2 transformed and had batch effects removed for final analysis.