Regulatory substances produced by lymphocytes : I. Inhibitor of DNA synthesis in the rat

A factor which suppresses DNA synthesis in rat lymph-node cells stimulated with phytohemagglutinin (PHA) and in mouse fibroblasts (L cells) was obtained from the culture supernatant of rat lymph-node cells stimulated with ovalbumin in vivo and rechallenged with the same antigen in vitro. The factor is protein in nature, as evidenced by its sensitivity to trypsin. It is relatively heat stable and its activity is lost after periodate treatment, suggesting that it is glycoprotein. On Sephadex gel filtration, it gives 2 peaks with estimated molecular weights of 80,000 and 160,000-200,000 daltons. Their isoelectric points were estimated as 2.7 and 3.0 by isoelectric focusing analysis. The factor had almost no effect on the synthesis of RNA and protein, but inhibited DNA synthesis completely. This inhibition followed multihit kinetics with a multiplicity estimated as 20-40. The suppressive effect of the factor was reversible when it was removed within 14 h after addition of PHA to cultures of rat lymph-node cells, but became completely irreversible by 30 h. Between 14 and 30 h the effect was partially reversible. These results suggest that the factor acts by inhibiting a process or processes occurring shortly before the initiation of DNA synthesis. They also imply that the factor may play a role as a short-range regulator of immune responses.