Allograft Inflammatory Factor 1 as an Immunohistochemical Marker for Macrophages in Multiple Tissues and Laboratory Animal Species

Abstract

Allograft inflammatory factor 1 (AIF1) is a commonly used marker for microglia in the brains of humans and some animal models but has had limited applications elsewhere. We sought to determine whether AIF1 can be used as a macrophage marker across common laboratory animal species and tissues. We studied tissues (that is, spleen, liver, and lung) with defined macrophage populations by using an AIF1 immunostaining technique previously validated in human tissue. Tissues were collected from various mouse strains (n = 20), rat strains (n = 15), pigs (n = 4), ferrets (n = 4), and humans (n = 4, lung only). All samples of liver had scattered immunostaining in interstitial cells, consistent with resident tissue macrophages (Kupffer cells). Spleen samples had cellular immunostaining of macrophages in both the red and white pulp compartments, but the red pulp had more immunostained cellular aggregates and, in some species, increased immunostaining intensity compared with white pulp. In lung, alveolar macrophages had weak to moderate staining, whereas interstitial and perivascular macrophages demonstrated moderate to robust staining. Incidental lesions and tissue changes were detected in some sections, including a tumor, inducible bronchus-associated lymphoid tissue, and inflammatory lesions that demonstrated AIF1 immunostaining of macrophages. Finally, we compared AIF1 immunostaining of alveolar macrophages between a hypertensive rat model (SHR strain) and a normotensive model (WKY strain). SHR lungs had altered intensity and distribution of immunostaining in activated macrophages compared with macrophages of WKY lungs. Overall, AIF1 immunostaining demonstrated reproducible macrophage staining across multiple species and tissue types. Given the increasing breadth of model species used to study human disease, the use of cross-species markers and techniques can reduce some of the inherent variability within translational research.

Figure 1.

(A through C) AIF1 immunostaining (brown) in tissues and (D) scoring. (A) In liver, AIF1 immunostaining is seen in scattered interstitial cells consistent with Kupffer cells. (B) In lung, AIF1 immunostaining is moderate to robust in scattered interstitial macrophages, with weak to moderate staining in alveolar macrophages. (C) In spleen, AIF1 immunostaining in macrophages is prominent in red pulp, with fewer stained cells in the white pulp. DAB chromogen and hematoxylin counterstain; magnification, 100×. (D) Ordinal scoring and Wilcoxon matched-pairs signed rank analysis of AIF1 immunostaining intensity in splenic macrophages reveals more intense staining in red pulp than white pulp in mice (n = 20, P = 0.0002) and rats (n = 15, P < 0.0001); but differences in ferrets (n = 4) and pigs (n = 4) did not achieve statistical significance.

Figure 2.

AIF1 immunostaining (brown color) in (A through C) lung and (D) liver. (A) Several AIF1⁺ macrophages (arrows) within a papillary adenoma of a mouse lung. (B) Several AIF1⁺ cells (arrows) marginated along the vessel wall in a rat lung. (C) Scattered AIF1⁺ macrophages (arrows) within inducible bronchus-associated lymphoid tissue localized at the junction of a branching airway in a rat lung. (D) Several AIF1⁺ macrophages (arrows) in a chronic lesion of a rat liver. DAB chromogen and hematoxylin counterstain; magnification: 200× (A through C), 100× (D).

Figure 3.

AIF1 immunostaining and scoring. (A and B) Immunostaining of AIF1⁺ macrophages in human lungs that (A) lacked inflammation or (B) had focal inflammation shows enhanced AIF1⁺ macrophages within and around airspaces. (C and D) Lung samples from (C) WKY rats had alveolar macrophages with relatively uniform moderate cytoplasmic AIF1 immunostaining. In contrast, (D) SHR alveolar macrophages were larger, with preferentially eccentric cytoplasmic AIF1⁺ immunostaining. DAB chromogen and hematoxylin counterstain; magnification: 200× (A), 100× (B), 400× (C and D). (E) Semiquantitative rank scores for features of macrophage activation (for example, enlarged nucleus, increased foamy cytoplasm), P = 0.028 (unpaired t-test) between rat lines. (F) Quantitative scoring of macrophage diameter, P = 0.0016 (unpaired t-test) between rat lines.