Inhibition of Lymphotoxin- and Lymphocyte-Mediated Cytolysis by High Molecular-Weight Dextran 1

Abstract

Lymphotoxin (LT) is a cytotoxin released by antigen- or mitogen-stimulated lymphocytes in vitro (1). The inhibition of lymphocyte-mediated cytolysis by anti-LT serum (2), as well as other lines of evidence (1), suggest that this lymphokine has a role in lymphocyte-mediated cytotoxicity in tissue culture. Recently, Henney (3) and Ferluga and Allison (4) reported the protective effect of dextrans on lymphocyte-mediated target-cell destruction.

In this study, we examined the effect of high molecular-weight dextran (83,000) on lymphotoxin-induced cytolysis in tissue culture and compared our findings with those observed with dextran in cell-mediated cytolysis in vitro.

Materials and assays, target cells. Mono-layers of mouse L fibroblasts, clone 929, grown for 24 hr in round-bottomed tubes, were used as targets. The tissue culture medium was RPMI 1640 with 5% fetal calf serum (FCS).

Lymphotoxin. Human lymphotoxin was purified from supernatants of phytohemag-glutinin (PHA)-stimulated human adenoidal lymphocytes as previously described (5). The activity of LT is expressed in cytotoxic units (CU) as formerly defined (5). Lymphotoxin was dissolved in a buffer consisting of 0.1 M glycine-0.15 M NaCl, pH 9.5.

Spleen cells. Sensitized spleen cells were obtained from BALB/c mice which had received intraperitoneal and, 3-4 weeks afterward, intrasplenic injections of living L cells, derived from a C3H mouse as previously described (6). Spleens were harvested 3-6 days later; RBCs were lysed in hypo-tonic solution, and the spleen cells were resuspended in tissue culture medium. Control spleen cells were taken from nonin-jected BALB/c mice.

Dextran solutions. Dextran, average mol wt 83,000 (Sigma Chemical Co., St. Louis, Mo.), was dissolved in RPMI 1640 and di-alyzed against this medium. After dialysis, the concentration of dextran was adjusted to the desired molarity by adding RPMI 1640. Then, the material was filtered through Mil-lipore membranes (pore size, 0.22 μm), and FCS was added to yield a final concentration of 5% serum.