Visualizing cytokine-secreting cells in situ in the rhesus macaque model of chronic gut inflammation

Abstract

Cytokine-producing cells in gut-associated lymphoid tissues of rhesus macaques with chronic enterocolitis were studied. The confocal microscopy technique that we developed enables simultaneous in situ visualization of multiple extra- and/or intracellular antigens at a resolution higher than that allowed by light or epifluorescence microscopy. The presence of interleukin-6 (IL-6)-, tumor necrosis factor alpha (TNF-alpha)-, and IL-1-alpha-producing cells was focally intense in the colon lamina propria of the affected animals. The IL-1-alpha-producing cells were T lymphocytes (CD3+), while the TNF-alpha-producing cells were both macrophages (CD68+/HAM56+/LN5+) and T lymphocytes (CD3+). The IL-6-producing cells within the colon consisted of T lymphocytes and macrophages. The amount of IL-6-producing cells seen in macaques with enterocolitis was significantly higher (P<0.001) than that seen in the healthy control animal, while TNF-alpha- and IL-1-alpha-producing cells were seen only in macaques with enterocolitis. Most of the T lymphocytes that produced cytokines were detected in the lamina propria, while the macrophages were most prominent in highly inflamed regions of the lamina propria. Taken together, our findings indicate that there might be immunological similarity between chronic enterocolitis of rhesus macaques and humans, suggesting the potential use of the nonhuman primate model for the validation of novel therapies.

Figures

FIG. 1.

Multilabel confocal microscopy of cytokine-producing macrophages. (A) Multilabel confocal microscopy of cytokines (TNF-α and IL-6) and macrophages (LN5) in the colon lamina propria of a macaque with chronic colitis. Numerous IL-6-producing cells (red) and scattered TNF-α-producing cells (green) are present. The macrophages that are present (blue) are often positive for IL-6 (colocalized red and blue seen as pink). Autofluorescent bodies are seen as orange. (B) The colon lamina propria from a macaque with colitis showing an aggregate of TNF-α-producing cells. The LN5+ macrophages are producing IL-6 but not TNF-α. (C) Confocal microscopy for TNF-α, IL-6, and LN5 combined with a differential interference contrast (DIC) image of colon from a healthy control rhesus macaque. No TNF-α production was detected. Scattered LN5+ macrophages (blue) and IL-6-producing cells (red) are present, as are occasional IL-6-producing LN5+ macrophages (which appear pink). (D) Double-label confocal microscopy of the colon lamina propria of an animal with colitis showing TNF-α (green) and the macrophage marker HAM56 (blue). Macrophages producing TNF-α appear blue-green. (E) Confocal microscopy for TNF-α (green) and the macrophage marker CD68 (red) combined with a differential interference contrast image of a colon from a rhesus macaque with chronic enterocolitis. Several TNF-α-producing macrophages are present and appear yellow.

FIG. 2.

Multilabel confocal microscopy of cytokine-producing T lymphocytes. (A) IL-6 production by T cells: confocal microscopy for IL-6 (green) and CD3 (blue) combined with a differential contrast image of a colon lamina propria from a rhesus macaque with chronic enterocolitis. Numerous IL-6-producing T cells (blue-green) are present. (B) IL-6 production by T cells from the control animal: IL-6-producing cells are less common than those seen in the colitis tissues. (C) IL-1-α production by CD3+ T cells (blue-green) in the colon lamina propria of a rhesus macaque with colitis. (D) Confocal microscopy and DIC image of IL-1-α and CD3 in the colon lamina propria of a normal (control) rhesus macaque confirming the absence of IL-1-α in this tissue.