engleski

Localization of putative receptors for thymic parvalbumins of chickens to T cell regions of spleen and caecal tonsil

The chicken thymus produces two calcium-binding parvalbumins. Avian thymic hormone (ATH) is produced by epithelial cells in the thymic cortex, and it circulates in blood where it cycles every fifth day. It is apparently involved in immune development since it stimulates precocious development of cellular immunity but not humoral immunity. Chicken CPV3 is a closely related molecule elaborated in the same region of thymus but it has not been determined that both parvalbumins are produced by the same cells. Since ATH modulates immune ontogeny and circulates in blood, and CPV3 probably does so as well, it was proposed there should be cells in the periphery that are affected by these parvalbumins, and thus have receptors for them. Newly hatched chicks were injected intracoelomically with 1 mg ATH in an effort to have all putative receptors occupied. Chickens were killed by decapitation 5, 10 and 20 minutes later, and spleen, caecal tonsils, bursa Fabricius, brain, and liver quickly removed, fixed, and prepared for electron microscopy (EM). Tissue sections were blocked with normal goat serum, then incubated with monoclonal antibody (mAb) for either ATH or CPV3, washed, and incubated with colloidal gold-labelled goat anti-mouse globulin. Spleen and caecal tonsils also were examined for ATH receptors by fluorescence microscopy. Frozen sections were incubated in the same way except the second antibody was conjugated with fluorescein. Frozen and formalin fixed, paraffin-embedded (FFPE) sections of spleen were examined by immunohistochemical methods to determine immunophenotype of the receptor cells. Antigen retrieval procedure of Shi et al. (Cell Vision 2:255-256, 1995) was used on FFPE sections. Sections were labelled for receptor as described, using for second antibody colloidal gold-labelled goat anti-mouse globulin. Then, sections were incubated with biotinylated mAb for chicken CD3, CD4, CD8, TCR-1(g/d) and TCR-2(a/b), rinsed, and a mixture of avidin and biotin-alkaline phosphatase added. McGadey's reagent was used for formation of formazan by alkaline phosphatase. Sections were rinsed, and silver enhancing solution (Sigma) added. In sections examined by EM, grains of colloidal gold were found in thymus, as expected, and in T cell regions of spleen and caecal tonsils, but not in the other tissues. In spleen and caecal tonsils probed with mAb for ATH, grains appeared mostly on cell surfaces at 5 minutes, were mostly internalized at 10 minutes, and most of them were in the nucleus at 20 minutes. Label for CPV3 also was localized in the T cell regions and was present on plasma membranes, in cytoplasm, and in nuclei, but the loading experiment was not done to follow migration. The functional correlates of ATH and CPV3 in nuclei of these lymphocytes are unknown, but involvement in gene expression is an intriguing speculation. Localization of the receptor cells in T cell regions was clearly revealed by these staining techniques. In sections stained for immunophenotype, receptors for ATH were stained with sharp black granules and the lymphocyte markers with diffuse purple deposits. The receptor cells were positive for CD3, CD8, and the lymphocyte receptor TCR-1.

avian thymic hormone; receptors; T cells

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