Characterization of New Zealand White Rabbit Gut-Associated Lymphoid Tissues and Use as Viral Oncology Animal Model

Abstract

Rabbits have served as a valuable animal model for the pathogenesis of various human diseases, including those related to agents that gain entry through the gastrointestinal tract such as human T cell leukemia virus type 1. However, limited information is available regarding the spatial distribution and phenotypic characterization of major rabbit leukocyte populations in mucosa-associated lymphoid tissues. Herein, we describe the spatial distribution and phenotypic characterization of leukocytes from gut-associated lymphoid tissues (GALT) from 12-week-old New Zealand White rabbits. Our data indicate that rabbits have similar distribution of leukocyte subsets as humans, both in the GALT inductive and effector sites and in mesenteric lymph nodes, spleen, and peripheral blood. GALT inductive sites, including appendix, cecal tonsil, Peyer's patches, and ileocecal plaque, had variable B cell/T cell ratios (ranging from 4.0 to 0.8) with a predominance of CD4 T cells within the T cell population in all four tissues. Intraepithelial and lamina propria compartments contained mostly T cells, with CD4 T cells predominating in the lamina propria compartment and CD8 T cells predominating in the intraepithelial compartment. Mesenteric lymph node, peripheral blood, and splenic samples contained approximately equal percentages of B cells and T cells, with a high proportion of CD4 T cells compared with CD8 T cells. Collectively, our data indicate that New Zealand White rabbits are comparable with humans throughout their GALT and support future studies that use the rabbit model to study human gut-associated disease or infectious agents that gain entry by the oral route.

Keywords: gut-associated lymphoid tissue (GALT); human T cell leukemia/lymphoma virus 1 (HTLV-1); intraepithelial lymphocytes; lamina propria lymphocytes; lymphoid tissue; rabbit; review; small intestine.

Figure 1

Analysis of lymphocyte populations from different lymphoid organs in the rabbit gastrointesional tract by flow cytometry. The percentages of lymphocytes expressing CD45, CD4, CD8, IgM, and CD11b are shown for 9 tissues. (A) Peripheral blood mononuclear cells. (B) Mesenteric lymph node. (C) Spleen. (D) Peyer's patch. (E) Cecal tonsil. (F) Ileocecal plaque. (G) Appendix. (H) Intraepithelial lymphocytes. (I) Laminar propria lymphocytes. Means and standard deviations were calculated based on results obtained from ten 12-week-old New Zealand White rabbits.

Figure 2

Lymphocyte distribution in the ileocecal plaque of the rabbit (original magnification 100x). Immunohistochemistry of the ileocecal plaque with antibodies against (A) CD3, (B) CD79a, (C) CD4, and (D) CD8. The structure of the ileocecal plaque is similar to that of a Peyer's patch. (A) Anti-CD3 stains T cell–rich parafollicular regions as well as scattered cells throughout B cell–predominant follicles, the submucosa, and mucosa. (B) Inductive sites are flask shaped, consisting of CD79a positive follicles under a narrower CD79a positive subepithelial dome region. There is a predominance of (C) CD4 cells to (D) CD8 cells in all GALT effector sites.

Figure 3

CD4 and CD8 T cell distribution in cecal tonsil of the rabbit (original magnification 100x). Immunohistochemistry of the cecal tonsil with antibodies against (A) CD4 and (B) CD8 T cells. (A) Within T cell parafollicular zones, the majority of the cells are CD4 positive. T cell zones contain smaller numbers of (B) CD8-positive cells compared with (A) CD4-positive cells.

Figure 4

Quantitative area analysis for Ki-67 in the Peyer's patch of the rabbit (original magnification 100x). Immunohistochemistry of the Peyer's patch stained with antibody against Ki-67. Positive staining identifies the germinal center. Formalin-fixed, paraffin-embedded tissue sections were stained with antibody and scanned (Aperio ScanScope XT; LeicaBiosystems). The cross-sectional area of the Peyer's patch, ileocecal plaque, cecal tonsil, and appendix were measured using the pen tool in Aperio ImageScope Viewer. The follicular region and germinal center (proliferation zone, green line) areas were outlined and calculated as a percentage of the total cross-sectional area (yellow line), as described in Dasso and colleagues 2000). The germinal center was defined as the area staining with Ki-67 (Dasso et al. 2000).

Figure 5

Comparison of germinal centers within inductive sites. Based on the percentage of positive Ki-67 lymphocytes in the follicular region area, the Peyer's patches, ileocecal plaque, cecal tonsil, and appendix were compared. The Peyer's patches have the smalllest percentage of germinal center tissue of all inductive sites.