Tissue markers of alveolar macrophage accumulations in inhaled drug development

Abstract

Alveolar macrophage accumulations in the lungs of rodents and dogs that have been exposed to inhaled new drug candidates is an important cause of attrition during preclinical drug development because it is very difficult to decide whether the accumulations are adverse or nonadverse. The expression of markers of alveolar macrophage endocytosis, alveolar macrophage activation and lung inflammation were investigated by means of reverse transcriptase-polymerase chain reaction, immunohistochemistry and in situ hybridisation in samples of formalin fixed paraffin embedded lungs from male rats. Messenger RNA levels of CD68, a marker of endocytosis, were increased 1.4 to 2.4-fold in lungs that contained alveolar macrophage accumulations. Cytoplasmic immunostaining related to the CD68 protein was increased 1.43 to 5.66-fold when the alveolar macrophage accumulations were numerous, large and associated with neutrophils and lymphocytes. In contrast, the immunostaining was not increased (range 0.67 to 1.21-fold) when only small and sparse alveolar macrophage accumulations that consisted entirely out of macrophages were present. Messenger RNA levels of resistin like molecule alpha and acidic mammalian chitinase – markers of wound healing (M2) activated macrophages – were found to be increased 3.9 to 8.2-fold and 1.8 to 2.4-fold respectively, in samples of lungs that contained alveolar macrophage accumulations. Messenger RNA levels of inducible nitric oxide synthase, a marker of classical (M1) activated macrophages was found to be increased 4.1-fold when neutrophils and lymphocytes were present amongst the accumulated alveolar macrophages. Messenger RNA levels of pro-inflammatory genes (such as E selectin) were found to be increased 2.2 to 4.4-fold in samples of lungs that contained alveolar macrophage accumulations but only when neutrophils and lymphocytes were visible amongst the accumulated macrophages in histological lung sections. Measuring the markers in FFPE lung samples can potentially help investigators discriminate adverse from nonadverse drug-induced alveolar macrophage accumulations with greater clarity and scientific rigour.