EFFECTS OF DIALYZABLE LEUKOCYTE EXTRACTS WITH TRANSFER-FACTOR ACTIVITY ON LEUKOCYTE MIGRATION INVITRO .2. SEPARATION AND PARTIAL CHARACTERIZATION OF THE COMPONENTS IN DLE PRODUCING ANTIGEN-DEPENDENT AND ANTIGEN-INDEPENDENT EFFECTS

  • 1 January 1979
    • journal article
    • research article
    • Vol. 93  (5) , 818-837
Abstract
Previous studies showed that DLE [dialyzable leukocyte extracts] with TFd [dialyzable transfer factor] activity produce Ag[antigen]-dependent specific effects (meadiated by TFd) and Ag-independent effects on CMI [cell mediated immunity] as demonstrated in vitro by agarose LMI [leukocyte migration inhibition]. Sephadex G-25 gel filtration provided a simple method for separating the DLE components responsible for each effect into distinct fractions. Ag-independent LMI was produced predominantly by Sephadex fraction I, of MW > 5000. The active components, further purified on Bio-Gel P-10, were of MW 14,000-17,000 and contained polypeptide and ribonucleotide material. The Ag-independent LMI activity was stable to heating at 56.degree. C for 30 min but was partially destroyed at 80.degree. C for 30 min, and the responsible components acted on [human] PMN [polymorphonuclear neutrophils] directly. Ag-independent ELM [enhancement of leukocyte migration] was produced exclusively by material in Sephadex G-25 fraction V and acted directly on PMN, whereas the Ag-dependent specific LMI activity was predominantly in fraction IVb and to a lesser extent in fraction V and could not be detected in a direct assay using only PMN. A new activity, designated Ag-dependent ELM activity, which caused increased migration in the presence of Ag, was found in Sephadex fraction IVa. This latter activity might mask the Ag-dependent LMI activity in fraction IVb. Bio-Gel P-2 chromatography separated the components producing Ag-dependent and Ag-independent effects in fraction V into 2 separate subfractions (Va and Vb) of MW 1100-2000 and < 900. The activity in fraction IVb eluted at a position identical to the components in fraction Va on Bio-Gel P-2. Fractions Va and Vb contained polypeptide and ribonucleotide material. The Ag-dependent specific LMI or TFd activity was partially inactivated at 56.degree. C and completely destroyed at 80.degree. C. The components responsible for this TFd activity were further purified by HPLC [high-pressure reverse phase liquid chromatography] on ODS[octadecyl silane] resin. The TFd activity was mediated by components with retention times much greater than that of cyclic AMP. The active fraction was composed of polypeptide and ribonucleotide material but did not contain deoxyribonucleotides.