The pulmonary innate immune system in the respiratory tract eliminates inhaled pathogens. Several cell types contribute to host defense within a complex network. The aim of this study was to evaluate the role of macrophages during pneumonia and in the regulation of the epithelial response to microorganisms. We performed lung infection models in mice lacking myeloid RelA/p65. To study the mechanistic relationships between individual cell types, we applied co-culture models composed of airway epithelial cells (AECs) and macrophages. Mice lacking myeloid RelA/p65 showed significantly decreased bacterial clearance, cytokine expression and neutrophil influx. In addition, the induction of epithelial keratinocyte chemoattractant expression was blunted in these animals. In vitro, AECs were largely insensitive to ligands of Toll-like receptor (TLR)2 or TLR5. Exposure to secretory products of macrophages results in an increased release of pro-inflammatory cytokines and augmented antimicrobial activity. This was associated with increased expression of TLR genes and surface expression of the proteins. Experiments with blocking antibodies showed that the effect of macrophages depends on secreted mediators, including tumour necrosis factor-alpha. In conclusion, the present data show that myeloid RelA is critical for pulmonary host defense. One important mechanism is that macrophages induce the sensitivity of AEC's to microbial patterns.