ABSTRACT
The thymus is the site of production of mature T lymphocytes and thus is indispensable for the development and maintenance of the T cell-mediated arm of the immune system. Thymic production of mature T cells is critically dependent on an influx of bone marrow-derived progenitor T cells that undergo replication and selection within the thymus. Thymus cellularity and thymic hormone secretion reach a peak during the first year of life and then decline gradually until the age of 50-60 years, a process known as "thymic involution." A rapid reduction of thymus cellularity occurs in young patients following injuries, chemotherapy, and other forms of stress. The mechanisms underlying the involution process appear to be dependent on factors intrinsic to the thymic tissue, such as the local production of cytokines and chemoattractants, promoting the recruitment, growth, and differentiation of bone marrow-derived T cell progenitors in the thymus, as well as extrinsic factors, such as systemic levels of endocrine hormones and mediators released by intrathymic nerves of the autonomic nervous system. Knowledge of these factors provides a rational basis for the development of an approach based on tissue engineering that could be used to provide either temporary or permanent reconstitution of thymic function.
Subject(s)
Aging/immunology , Immunologic Deficiency Syndromes/therapy , T-Lymphocytes/immunology , Thymus Gland/growth & development , Thymus Hormones/metabolism , Animals , Antigens, Differentiation, T-Lymphocyte/analysis , Autonomic Nervous System/physiology , Cell Differentiation , Cell Lineage , Cell Movement , Cytokines/physiology , Hormones/physiology , Humans , Immunologic Deficiency Syndromes/etiology , Lymphocyte Count , Mice , Mice, Knockout , Mice, Nude , Mice, Transgenic , Stress, Physiological/immunology , T-Lymphocytes/drug effects , Thymus Gland/embryology , Thymus Gland/immunology , Thymus Gland/metabolism , Thymus Hormones/therapeutic useABSTRACT
Current fluorescence-based adhesion assays that use a 96-well plate format rely on the assumption that the fluorescent label does not significantly leak from the cells. Thus, we evaluated a calcein-based, in vitro adhesion assay in 96-well plates using five different types of leukocytes (HL60 cells, human neutrophils, rat neutrophils, mouse progenitor T cells and EL4 cells). Each cell type leaked calcein at a different rate, with the highest rates found for rat neutrophils and progenitor T cells, which lost as much as 20%-40% of the label within 90 min, the time required to complete the assay. Thus, we developed a procedure to measure the dye leakage rate during the assay in order to obtain a correction factor, which was then used to calculate the "true" number of adherent cells. Data for the adhesion of FTF1 cells to endothelial monolayers, after correction for calcein leakage, deviated less than 10% of adhesion data obtained with a well-established 51Cr-based assay.
Subject(s)
Cell Culture Techniques/methods , Fluorescent Dyes/metabolism , Neutrophils/physiology , T-Lymphocytes/physiology , Animals , Cell Adhesion , Fluoresceins/metabolism , HL-60 Cells , Humans , Mice , Rats , Spectrometry, FluorescenceABSTRACT
Recent in vivo studies suggest that tumor necrosis factor-alpha (TNF-alpha) is involved in the development of the thymus. We postulated that this inflammatory mediator could regulate the influx of progenitor T cells into the thymus. Using an in vitro static adhesion system, we found that TNF-alpha increases the adhesion of a murine progenitor T cell line (FTF1) to a bovine aortic endothelial cell line (1F8), human umbilical vein endothelial (HUVE) cells, and a murine arterial endothelial (MAE) cell line. TNF-alpha treatment of the 1F8 cells resulted in a time- and dose-dependent increase in the adherence of FTF1 cells. Adherence increased during the first 6 hr of treatment with TNF-alpha concentrations ranging from 10(-11) to 10(-9) M. Maximal adherence (6 hr treatment with 10(-10) M of TNF-alpha) was approximately 4.5-fold larger than that of untreated monolayers. A slow decrease in adherence, down to approximately 2-fold at 48 hr, was observed beyond 12 hr of TNF-alpha treatment; in contrast, removal of TNF-alpha after 6 hr of continued stimulation caused the adherence to return to pre-stimulation levels within 24-30 hr. Adhesion of FTF1 cells to TNF-alpha treated 1F8 cells was almost completely blocked by a monoclonal antibody against murine CD49d (very late antigen-4) expressed on FTF1 cells. TNF-alpha-induced adhesion of FTF1 cells to MAE cells was also blocked by monoclonal antibodies against murine CD49d and CD106 (vascular cell adhesion molecule-1). These results support the notion that local secretion of TNF-alpha could modulate the dynamics of adhesion of progenitor T cells to the thymic endothelium.
