003 1-3998/91/2903-0237$03.00/0 PEDIATRIC RESEARCH Vol. 29, No. 3. 1991 Copyright O 1991 International Pediatric Research Foundation, Inc. I'rinrcd in U.S. A. The Postnatal Development of Gut Lamina Propria Lymphocytes: Number, Proliferation, and T and B Cell Subsets in Conventional and Germ-Free Pigs1 H. J. ROTHKOTTER, H. ULBRICH, AND R. PABST Cenrre oJAnatomy. Medical School of Hannover, Hannover, Gcrtnany ABSTRACT. The lamina propria (LP) contains many lym- nutritional antigens increases when the mother's milk is gradually phocytes that are effector cells as well as memory cells of replaced by other forms of food (3). Therefore, major changes the gut immune system. This compartment was studied in can be expected in the number and subset composition of normal and germ-free pigs in the early postnatal period up lymphocytes in the LP. to 91 d of age. The number of LP lymphocytes nearly In this study, the LP lymphocytes were examined in pigs for doubled between the 1st and 29th d. LP lymphocytes several reasons: pigs can be reared under germ-free conditions proliferated more in the crypt region than in the villi with (10); being omnivorous, pigs have been used in experimental a mitotic rate/h comparable to that in nonfollicular com- gastroenterology for studying the development of intestinal en- partments of Peyer's patches and lymph nodes. The deter- zymes (1 1) as well as mucosal permeability (12); and pigs can mination of the subpopulations of LP lymphocytes showed serve as animal models for the Campylobacter jejuni infection a 10-fold increase in CD2+ cells between d 1 and 40. About (13). In the postnatal period in pigs, the size of the organized 80% of the LP T cells in 1- and 5-d-old pigs had the lymphoid tissue of the gut, the PP, increases dependent on unusual CD2+CD4-CD8- phenotype. Ig-positive cells ap- microbial and nutritional antigens, and the lymphocytes prolif- peared later in the postnatal period than the T cells. On d erate dependent on age (14). In the conventional pig, the lym- 1, only a few IgM+ cells were observed. In 40-d-old ani- phocyte subset composition shows major differences in the two mals, the number of IgA+ cells exceeded that of IgM'. Ten types of PP (15), which are absent in germ-free animals. times more Ig' cells were detected in the crypt region than Our study had the following aims: 1)to determine the number in the villi. The germ-free pigs at an age of 49 d had a T of LP lymphocytes at different ages in the postnatal period, 2) cell subset pattern comparable to that of 5-d-old normal to study whether lymphocytes proliferate locally in the LP, 3) to animals. (Pediatr Res 29: 237-242, 1991) determine the T and B subpopulations of LP lymphocytes to find out whether there are different patterns of development for Abbreviations each subset, and 4) to study the lymphocyte numbers and subsets under germ-free conditions to get an idea of how microbial LP, lamina propria antigen influences the development of the LP cells. PP, Peyer's patches TBS, Tris-buffered saline MATERIALS AND METHODS VCR, vincristine sulphate Animals. Normal and germ-free male and female German landrace pigs of different ages were examined. The animals had been reared in different ways as described earlier (14). The various groups and the different experiments camed out in these The gut immune system is an essential part of the bamer groups are listed in Table I. All animals were killed using an i.v. function of the gut wall. The uptake of microbial and nutritional injection of T 61 (embutramid, mebezoniumjodit, tetracainhy- antigens in the gut starts immune responses by initiating either drochlorid; Hoechst Veterinar, UnterschleilJheim/Munich, Ger- tolerance or a specific secretory immune reaction (for review see many). refs. 1-4). Large numbers of lymphocytes are distributed in the Lymphocyle prolferarion. These experiments had been done epithelium and LP of the gut mucosa. Some of the LP lympho- earlier to study lymphocyte proliferation in the PP (14, 16). Now cytes are PP-derived B lymphoblasts, becoming mature plasma tissue samples of the same pigs were used to determine prolifer- cells in the LP. They preferentially produce IgA (5, 6). The ation in the LP. In brief, pigs of 1, 5, 12, 40, and 91 d of age majority of T cells in the LP are CD4+ (7, 8). The T cells show were injected with 0.25 mg/kg body weight of VCR (Eli Lilly, a high expression of genes associated with cell activation (9). The Giessen, Germany) i.v. to arrest all mitoses in the metaphase. At proportion of antigen-primed memory cells is higher in LP T 1, 2, 2.5, or 3.5 h after the VCR injection, the animals were cells than in peripheral blood lymphocytes (reviewed in 4). killed. The 13-wk-old animals were laparotomized under anes- In the early postnatal period, the gut wall is exposed to micro- thesia (Thiopental: Trapanal, Byk-Gulden, Konstanz, Germany) bial antigens for the first time. Additionally, the amount of and in every pig samples of the gut were taken for histology Received April I 1, 1990; accepted October 9, 1990. before and 1, 2, 2.5, and 3.5 h after the injection of VCR. Correspondence: Dr. H. J. Rothkotter, Zentmm Anatornie 4150, Medizinische Histology and immunohistology. Pieces of the jejunum and Hochschule Hannover, Postfach 6 10 180, D-3000 Hannover 6 1, Germany. ileum were excised and fixed in Schaffer's solution. The tissue Supported by the Deutsche Forschungsgemeinschaft,"Gastrointestinal Banier," was embedded in glycolmethacrylate, cut at 4 pm and stained Project C I. ' Presented in part at the 5th lnternational Congress of Mucosal Immunology. with Giemsa or hematoxylin and eosin solution. For immuno- London, July 1989. histology the following method was used as described previously 2 Table 1. Animal groups and experimental design Number of Breeding Lymphocyte Lymphocyte animals conditions number Proliferation subsets Conventional Conventional Feeding machine Feeding machine Feeding machine Feeding machine Feeding machine Feeding machine Conventional Germ-free Table 2. MAb used in this study (1 7): Small parts of the gut were frozen in liquid nitrogen and Antibody Specificity Reference Dilution stored at -70°C. Cryostat sections (5 pm) were cut, dried for at least 1 h, and stored at -20°C. After being removed from the T cells freezer, the cryostat sections were dried for 30 min and then 74-12-4 CD4 18 1:200 fixed for 90 s in equal parts of methanol and acetone. TBS 295/33 CD8 18 1:30 containing 0.05% Tween 20 (Serva, Heidelberg, Germany) 8/ 1 Resting T cells 18 1 :400 served as washing buffer. The sections were incubated with 50 Mac80 CD2 18 1 :300 pL of the primary antibody dilution for 30 min in a humid Ig' cells chamber. The MAb (reviewed in ref. 18) used in this study are 27.9.1 anti-pig IgA 18 1 :20000 listed in Table 2. The sections were incubated with the second 23.7.1 b anti-pig IgG 18 1:20000 (rabbit anti-mouse Ig) and third antibody (alkaline phosphatase 28.4.1 anti-pig IgM 18 1:20000 anti-alkaline phosphatase complex), both diluted 150 (Dako, Hamburg, Germany). The color reaction was carried out with a fast red solution (10 mg fast red salt in 9.8 mL 0.1 M TBS containing 2 mg naphthol AS-MX phosphate, 200 pL N,N- dimethylformamide, and 10 pL 1 M levamisole, all from Sigma, Munich, Germany), counterstained, and mounted in glycergel (Dako). For sections of 12- and 40-d-old animals the indirect peroxidase method was used: goat anti-mouse F'(ab)2 fragments diluted 150 served as second antibody and the surface labeling was developed in these sections with 3,3'-diaminobenzidine- tetrahydrochloride (Sigma) dissolved in TBS at a concentration of 1 mg/mL containing 0.02% H202. These sections were coun- terstained as described above and mounted. To test whether the - o conventional animals sensitivity of both methods was comparable, in six samples both techniques were used. The number of positive cells/mm2 were , ' :::I similar (40-d-old animals, alkaline phosphatase or peroxidase 0 rea , , , , . , , , , technique: CD2+, 1340 f 240 and 1240 f 396; CD8+, 910 rt 0 10 20 30 40 50 60 70 80 90 270 and 780 & 290 cells/mm2). Evaluation. Only cells with all the morphologic characteristics Fig. I. Number of lymphocytes in the LP of the gut mucosa of of lymphocytes were counted. A Zeiss microscope was used with conventionally reared pigs of different ages and in germ-free animals 49 an ocular grid containing 100 rectangles (total area, 0.0 1 mm2 at d of age. The results are given as the mean number of cells/mm2 + SD. 625X magnification). The cell counts were carried out as follows: The grid was moved in the villi or crypt region of the jejunum and ileum. At 625x magnification, the area of the LP was determined by counting the number of the rectangles placed over the LP. The number of lymphocytes, mitoses, or membrane- stained lymphocytes in the measured area was counted. The cell number/mm2 was calculated with the number of the counted EE4 villi 0crypts rectangles. For the number of lymphocytes and mitoses, at least 50 grids and for lymphocyte subsets at least 20 grids were examined both in the crypt and in the villi region. Using regression analysis, the relative number of mitoses per h was calculated, and p 5 0.05 in the two-tailed t test for linear regression was regarded as significant for the relative number of mitoses per h.