Tumor necrosis factor-α binding in porcine primary stromal-vascular cell cultures

The binding characteristics of tumor necrosis factor-a receptors (TNFRs) in primary stromal-vascular cultures from fat tissue of 7-d-old pigs were analyzed. Cells were plated and maintained in 10% fetal bovine serum from day 0 to day 3 and then switched to serum-free medium from day 3 to day 6 to induce lipid filling. On days 3 and 6 of culture, some of the cells were lysed for ligand and immunoblotting and the remainder subjected to competitive and inhibitory-binding assays. Media from day 6 of culture were subjected to ligand and immunoblotting. Competitive binding analysis showed one-site bindings, with IC(50)s in the nanomolar and K(d)s in the picomolar ranges, that were not significantly different at both time-points of measurement. However, the B-max decreased significantly with differentiation. Preincubation with antibody against TNF receptor type 1 (TNFR1) or TNF receptor type 2 reduced the specific binding by 95 and 15%, respectively, suggesting a dominating role of TNFR1 in (125)-labeled TNF alpha ((12.5)-TNF alpha) binding. This was further supported by ligand blotting of cell lysates. Ligand and immunoblotting of cell lysates indicated that TNF alpha utilizes both types of surface receptors and their isoforms which were not modified during differentiation. Ligand blotting of media revealed soluble receptors with high M-x implying the formation of multimers. Immunoblotting suggested the presence of both types of TNFRs, but a greater abundance of soluble TNFR1. Also, it indicated the additional formation of smaller oligomers from both types of soluble receptors suggesting higher affinity of larger multimers for I-125-TNF alpha.