Quantification of proliferating lymphocyte subsets appearing in the intestinal lymph and the blood

Lymphocyte emigration from the intestinal wall via lymphatics is necessary to maintain gastrointestinal immunity and also to connect the different parts of the mucosal immune system. In the present study the numbers and time kinetics of proliferating lymphocyte subsets leaving the gut wall via intestinal lymphatics were analysed in mesenteric lymph node adenectomized minipigs (n = 8). After cannulation of the major intestinal lymph duct, afferent lymph was collected under non-restraining conditions. In four pigs lymphocytes taken from the intestinal lymph and blood were incubated in vitro with the thymidine analogue bromodesoxyuridine (BrdU) to label all lymphocytes in the S-phase of the cell cycle. The other four pigs received a single i.v. injection of BrdU 1 week after cannulation. The initial percentage of BrdU+ lymphocyte subsets in the intestinal lymph 15 min after BrdU injection was comparable to that after the in vitro labelling (1.5 +/- 0.7% in T cells, 10.6 +/- 1.6% in IgM+ cells and 30.0 +/- 11.9% in IgA+ cells). From this level onwards, the percentage of in vivo labelled BrdU+ lymphocyte subsets reached a maximum at 12 h after BrdU application. A different pattern of BrdU+ subsets was seen in the blood. After an early peak at around 3-4 h, the frequency of BrdU in vivo labelled cells decreased. Each subset had a maximum between 12 h and 48 h after BrdU application (maximum of BrdU+ CD2+ T cells at 12 h, 4.6 +/- 1.5%; IgM+ BrdU+ at 48 h, 8.8 +/- 3.3%). The present results provide a basis to determine the time necessary for induction of specific intestinal immunity during oral vaccination studies.