Preliminary Characterization of IF Inhibitory Activity
The T cell-inhibiting activity of IF was stable at 60°C (up to 30 min) but was reduced by heating at 100°C for 5 min (Fig. 5A). These observations were not altered when the samples were dialyzed after heat treatment. Some stimulatory activity, however, continued to be detected in IF samples treated at 100°C. The IF inhibitory activity was not affected by dialysis under acid (pH 2.0) conditions (Fig. 5B).
The majority of the inhibitory activity in IF was retained in the large (> 100 000) molecular weight fraction after ultrafiltration, with a minor amount of activity (approximately 10-20% of total) passing through in the small (< 10 000) molecular weight fraction (Fig. 5C). Stimulatory activity was found in the intermediate ultrafiltration fraction of IF, consistent with the molecular size of IL-1. The inhibitory activity of IF also was reduced by incubation with trypsin (Fig. 5D). buy ortho tri-cyclen
Comparison of IF and TGFfil showed very different dose-response curves in the rat thymocyte assay (Fig. 6). Coincubation with a polyspecific TGF|3 antiserum directed against all three mammalian TGFps inhibited TGF(31 but had no effect on IF (Fig. 7).
FIG. 5. Biochemical characteristics of inhibitory activity. [3H]Thymidine incorporation by rat thymocytes in the absence of stimulation (basal), with PHA alone, and with PHA in the presence of IF after various experimental treatments. A) Dose-equivalent (5 |xl) of normal rat testicular IF before (SM) and after heating at 60°C (10 min) or 100°C (5 min). B) Dose-equivalent (5 (x!) of normal rat testicular IF before (SM) and after dialysis at pH 7 or pH 2. C) Dose-equivalent (10 (J) of normal rat testicular IF before ultrafiltration (SM) and after fractionation into < 10 000 (< 10), 10 000-100 000 (> 10), and > 100 000 (> 100) molecular weight fractions. D) Dose-equivalent (1.25 ^l) of normal rat IF following incubation with heat-inactivated trypsin (0 min) or active trypsin at 37°C (60 min). Values are mean ± SEM, n = 4 replicate wells. Values with same letter superscript are not significantly different (p < 0.05).
FIG. 6. Comparison of the dose-response effects of normal rat testicular IF and TGFp$1 on PHA-induced [3H]thymidine incorporation in the rat thymocyte proliferation assay. Values are mean ± SEM, n = 4 replicate wells. Comparisons are with the control (PHA alone) response: *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significantly different (p > 0.05).
FIG. 7. [3H]Thymidine incorporation by rat thymocytes with PHA alone, with PHA and IF (5 jxl), or with TGF0 (4 ng/ml) in the absence (open bars) or presence (solid bars) of a polyspecific TGFp antiserum (80 (jug/ ml). Values are mean ± SEM, n = 4 replicate wells. Values with same letter superscript are not significantly different (p < 0.05).