Perforin expression in the gastrointestinal mucosa is limited to acute simian immunodeficiency virus infection

Abstract

Perforin-mediated cytotoxicity is a major effector function of virus-specific CD8 T cells. We have investigated the expression of perforin in the gut, an important site of simian immunodeficiency virus (SIV) pathogenesis, during experimental SIV infection of rhesus macaques. We observed significant increases in perforin protein and mRNA expression levels in the colons of SIV-infected macaques as early as 21 days after infection. However, during chronic infection, despite ongoing viral replication, perforin expression returned to levels similar to those detected in SIV-naïve animals. These findings demonstrate the presence of a robust perforin-positive response in gastrointestinal CD8 T cells during acute, but not chronic, SIV infection.

FIG. 1.

Perforin expression in colon and spleen in SIV-positive and -naïve macaques. Immunohistochemical staining of colon and spleen tissues from macaques indicates various levels of perforin expression between SIV-naïve animals and SIV-positive animals at 29 and 180 days postinfection (PI). The frequencies of CD8⁺ cells can be observed in sequential sections. Perforin and CD8 are stained brown by diaminobenzidine, and cell nuclei are stained blue by hematoxylin.

FIG. 2.

Quantitative assessment of perforin protein and perforin mRNA in colon and spleen tissues from SIVmac251-infected macaques. For analysis of perforin expression in tissues by immunohistochemical staining, each total perforin-positive area was measured and expressed as a percentage of the total cell area (A and B). Changes in perforin mRNA are reported as increases (n-fold) of perforin mRNA levels in SIV-infected colon (C) and spleen (D), compared to that in SIV-negative (SIVneg) animals (n = 8) (1). If statistical differences existed between groups as determined by a one-way analysis of variance, then a Holm-Sidak test was used to determine significant differences between pairs of groups, and all P values are indicated in the figure. Necropsy times shown in the figure are averages for each group of animals; for actual necropsy times for individual animals, see Table 1.

FIG. 3.

Immunofluorescent staining and confocal microscopy of colon and spleen tissues reveal expression of perforin in CD8⁺ cells. On day 21 postinfection (PI), animal 28240 shows perforin (green) expressed in CD8⁺ cells (red). Cell nuclei are stained with 4′,6′-diamidino-2-phenylindole, which appears blue, and colocalization of perforin with membrane CD8 appears yellow. Images were obtained using a Leica TCS-SP2 confocal microscope. Bars, 20 μm.