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For induction of TGF-β 1 , spleen cells were cultured in serum-free medium X-Vivo-20 (BioWhittaker, Inc., Walkersville, MD, USA) in the presence of proteoglycan.
THP-1 (generously provided by Dr P Guyre, Dartmouth Medical School), Jurkat (ATCC), and 293 cell lines were cultured in RPMI complete medium (CM), consisting of RPMI 1640 with 10% heat-inactivated fetal bovine serum, 0.1 ml of sodium pyruvate, 2.0 mmol/l of L-glutamine, 25 mmol/l of 1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 0.1 mmol/l of nonessential amino acids, 0.25 μg/ml of amphotericin, 50 μg/ml gentamicin (all from BioWhittaker, Walkersville, MD, USA) and 5 × 10 -5M 2-mercaptoethanol (Sigma, St Louis, MO, USA).
Cells were cultured in DMEM/F-12 medium (Sigma) supplemented with 20% (v/v) FCS (Biowhittaker Inc., Walkersville, MD) under 5% CO 2 in air at 37°C.
Purified bovine CL-derived microvascular endothelial cell populations were generated commercially by Cambrex Biosciences (BioWhittaker, Walkersville, MD) as described in detail [ 35 ] . In the present study, CLENDO cells from frozen aliquots (passage 3) were seeded (5,000 cells/cm 2) in 6- or 24-well plates or 10 cm culture dishes.
CV-1 cells were maintained in 10% fetal bovine serum (Bio-Whittaker, Walkersville, MD), 100 u/ml penicillin, 100 μg/ml streptomycin in DMEM.