Although immune system development seems partly regulated by internal triggers, its complete maturation appears to occur only after the intestine is challenged postnatally with both microbial and nutritional antigens. The aim of the present study was to investigate the immunoglobulin (Ig) secretion ability of cells from gut-associated lymphoid tissue (lamina propria lymphocytes, LPL) and spleen during the suckling period of Lewis rats.
The study was in compliance with the guidelines for the care and use of laboratory animals approved by the Ethic Commission for Animal Experimentation of the University of Barcelona. Spleen and small intestine were removed from decapitated rats aged between 1 and 21 days, and also from 9-week-old animals to be used as controls. Splenic cells were obtained by pressing the spleen through stainless-steel sieves and isolation with a Nycoprep gradient. To obtain LPL, the small intestine was treated with DTT-EDTA to remove intraepithelial lymphocytes. Then, the remaining tissue was enzymatically treated and cells were purified by Percoll gradient. Splenic cells and LPL were assayed for the spontaneous secretion of IgA, IgM, and IgG by enzyme-linked immunospot techniques (ELISPOT) using specific mouse anti-rat isotypes monoclonal antibodies (MoAb) coated to nitrocellulose plates. Cell suspensions were incubated for 20 h and later, Ig secretion was evaluated by subsequent addition of biotinylated anti-isotype MoAb, extravidin-peroxidase, and a peroxidase substrate. Spots were counted by an ELISPOT reader.
In spleen, a few IgG-secreting cells could be quantified along the suckling period and they decreased in adult rats. The number of IgM-secreting cells increased in spleen during the first 3 weeks of life, and diminished in adult rats. The number of IgA-secreting cells rose throughout the suckling period until adult age. The study in LPL revealed that there was no Ig production until day 14. From then, IgM and IgA production could be quantified. The number of IgM-secreting cells was higher than that of IgA from day 14 to day 21. In contrast, in adults, intestinal IgA-secreting cells were more abundant than IgM-secreting cells. Gut spontaneous production of IgG was not observed.
In conclusion, we observed an earlier Ig secretion in spleen than in LPL, the splenic IgG and IgM secretion being higher during the suckling period than in adults rats. With regard to LPL, during the first weeks of life, the IgM secretion prevailed over IgA secretion but this proportion shifted in adult rats which would correspond to the main levels of IgA in intestinal lumen.
This work was supported by the Ministerio de Educación y Cultura (AGL-2000-0913).