Aging Aging

284 BeckmanT-cell adherence to vascular endothelium is, I believe, an important issue inimmunogerontological studies. To egress the circulation and enter tissues,T-cells must interact with, and adhere to, endothelial cells. Changes in T-cell–endothelium interactions may well have implications for atherogenesis andthe observed age-related decline in cell-mediated immunity, particularlydelayed type hypersensitivity (DTH). For example, activated “old” T-cells maydisplay an increased propensity to “stick” to endothelial cells (EC) and facilitateinflammation, or conversely, they may exhibit a diminished ability to bindand transmigrate through the endothelium. To this end, I have included in thischapter a simple assay that provides a measure of the capacity of variousT-cells to bind human umbilical vein endothelial cells.2. Materials1. Complete culture medium (CCM); RPMI-1640 (Flow Labs) supplemented with10% heat-inactivated fetal calf serum (FCS), 2 mM glutamine (added just prior toculture) and penicillin/streptomycin (10,000 U/mL) (see Note 1).2. Sterile flat, 96-well microtiter (Linbro) and 24-well tissue culture (Costar) plateswith lids (see Note 2).3. Phosphate buffered saline (PBS).4. 50 mM Tris-HCl, pH 9.5.5. Tritiated thymidine ([ 3 H]-TdR) (Amersham).6. Exogenous activators: MAbs (OKT3 [anti-CD3] [from American Type CultureCollection], 64.1[anti-CD3] and 9.3 [anti-CD28] [from Bristol–Myers–Squibb,Seattle], T112 and T113 [anti-CD2] [from E. Reinherz, MIT, Boston MA, USA]);PHA (Sigma), phorbol 12-myristate 13-acetate (PMA) (Sigma), SEB (SEB)(from Sigma), fibronectin (Collaborative Research Incorporated, or IntegratedSciences), and various cytokines (Boehringer Mannheim and Genzyme).7. Goat anti-mouse IgG (GAM) (Dakopatts).8. Mitomycin C (Sigma).9. Collagenase (Sigma).10. Fluorescein-labeled (FITC-) goat or sheep anti-mouse IgG (Becton-Dickinson).11. Vanyl ribonucleoside complexes (Gibco-BRL).12. 123-basepair DNA ladder (Gibco-BRL).13. Endothelial cell growth factor supplement (Integrated Sciences).14. ECV304: Transformed immortal human endothelial cell line from umbilical cord(ATCC CRL-1998).15. Trypsin/EDTA solution (1×) in PBS (Boehringer Mannheim).3. Methods3.1. T-Cell Activation Using Unfractionated PBL1. Add 20 µL or 100 µL of MAb OKT3, at a single predefined optimal concentration(e.g., 50 ng/mL; note the final concentration in each well is 5 ng/mL) or use