Abstract
Using the Toponome Imaging System (TIS), a serial immunostainer, we studied the patterns of expression of multiple markers in alveolar macrophages (AM) from female mice lacking surfactant protein A (SP-A knockouts; KO) after "rescue" with exogenous SP-A1. We also used a 7-marker subset to compare with AM from males. AM were harvested 18 h after intrapharyngeal SP-A1 or vehicle, attached to slides, and subjected to serial immunostaining for 12 markers. Expression of the markers in each pixel of the image was analyzed both in the whole image and in individual selected cells. The marker combination in each pixel is referred to as a combinatorial molecular phenotype (CMP). A subset of antibodies was used to compare AM from male mice to the females. We found: (a) extensive AM heterogeneity in females by CMP analysis and by clustering analysis of CMPs in single cells; (b) AM from female KO mice respond to exogenous SP-A1 by increasing CMP phenotypic diversity and perhaps enhancing their potential innate immune capabilities; and (c) comparison of male and female AM responses to SP-A1 revealed that males respond more vigorously than females and clustering analysis was more effective in distinguishing males from females rather than treated from control.