Jim Klostergaard

Human and murine lymphotoxins as a multicomponent system: Progress in purification of the human αL component

Abstract Lymphotoxins derived from activated lymphocytes from human and murine lymphoid cells are heterogeneous with respect to molecular size and charge, as well as with respect to the expression of carbohydrate residues. These molecules form a system of interrelated subunits, as evidenced by their shared antigenic determinants, as well as by the reversible dissociation of the smaller forms from the larger. Although the smaller molecular weight forms (α l , β, γ) are apparently only capable of relatively protracted lysis of selected strains of the murine L-929 cell, the higher molecular weight forms (Cx, α H ) appear to be capable of rapid lysis of the L cell, as well as of relatively rapid, nonspecific lysis of other cells. Furthermore, the Cx forms appear to be associated with an antigen binding receptor which may be of T cell origin. Moreover, these forms released by alloimmune murine T cells can specifically lyse allogeneic tumor cells used in sensitization. The human Cx and α H LT also appear to express determinants encoded by genes of the MHC. Presently, we have been able to incorporate 125 I into both human and murine lymphotoxin preparations, while fully preserving biological activity. This has enabled us to monitor our attempts at purification of these materials through several consecutive isolation procedures including molecular sieving, ion-exchange chromatography, lectin affinity chromatography, hydrophobic chromatography and electrophoresis. Our results indicate that these materials are present in lymphocyte supernatants in extremely small amounts, probably less than 25 ng/ml; thus the purification of each component by biochemical techniques will require very vigorous methods.

Purification of murine macrophage cytotoxin (MCT).

Macrophage cytotoxin (MCT) can be induced from peritoneal exudate macrophage monolayers (PEMM) obtained from thioglycollate‐treated mice, by exposure of PEMM to lipopolysaccharide (LPS) or to double‐stranded polyinosinic: poly‐cytidylic acid (poly‐l:poly‐C). MCT is highly labile even upon storage at 4°C, and is irreversibly denatured by isolectricfocusing, polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (SDS), or by exposure to ethylene glycol. α‐MCT [150,000 daltons (d)] has been highly purified (2,000‐ to 5,000‐fold) from serum‐free, PEMM supernatants by a scheme of concentration, molecular sieving on Ultrogel AcA 44, negative hydrophobic affinity chromatography on benzyl‐agarose, and ion exchange chromatography on aminoethyl‐agarose. The scheme results in high yield of MCT, in part because of the rapidity with which the labile toxin is manipulated due to the tandemization of the chromatographic steps.

Purification of human alpha-light class lymphotoxin to electrophoretic homogeneity

Abstract We have purified to homogeneity the α L -component (70,000–90,000) of the human LT cytolytic system. This lymphokine was purified ~ 10, 000-fold from supernatants of lectin-stimulated human tonsil and adenoid lymphocytes by molecular sieving, ion-exchange chromatography on DEAE-Sepharose, and preparative PAGE. The homogeneity of the radiolabeled molecule was confirmed both by electrophoresis and electrofocusing. The identity of the labeled peak with the lytic activity was demonstrated with a concomitant bioassay of the electrophoresed preparation; in addition, immunoprecipitation with a heterologous specific anti-α L antiserum showed simultaneous precipitation of the radiolabeled component and lytic activity. Immunological and biochemical evidence has previously shown this molecule to be a subunit of the Cx- and α H -LT forms. The latter LT classes are of intense interest because of their capacity for rapid selective cell lysis.

The use of protein A in solid-phase binding assays: a comparison of four radioiodination techniques☆

Preparations of protein A radioiodinated by 4 different methods have been compared in indirect radioimmunoassays. The oxidative methods (chloramine-T and iodogen) for direct iodination of tyrosyl and histidyl residues were applied with high efficiency and gave a suitable product, provided the substitution ratio was kept low (1 iodine atom/molecule of protein A). Higher levels of modification tended to perturb the Fc-binding characteristics of the protein, especially with the use of iodogen. Introduction of the isotope via substitution of lysyl residues (Bolton-Hunter and Wood reagents) was also examined. The Bolton-Hunter modification of protein A gave an unsuitably low labeling efficiency; in contrast, the Wood reagent gave efficiencies approaching 50%. Protein A could be extensively substituted with the latter reagent (greater than 5 diiodinated benzimidate molecules per protein molecule). Thus, the use of the Wood-labeled protein A could raise the sensitivity of the binding assay at least an order of magnitude compared to using protein A iodinated by the oxidative methods. The effects on the biological activity of protein A exerted by the different labeling procedures are rationalized on the basis of the amino acid composition and tertiary structure of the protein.

Lymphotoxins — A Multicomponent System of Growth Inhibitory and Cell-Lytic Glycoproteins

Activated lymphocytes from experimental animals and man can release materials, termed lymphotoxins , which cause growth inhibition and cell lysis in vitro. These molecules, from human lymphocytes, are glycoproteins which can be divided into five molecular weight classes. These forms are heterogeneous, for each MW class can be further subdivided into multiple charge subclasses. It is now clear certain MW classes are interrelated and form a system of cell toxins. The larger classes (greater than 140,000 d) are associated with nonclassical antigen-binding receptors (R), which can be of T cell origin. The smaller forms (less than 90,000 d) do not express R function and are derived from the larger forms, possibly by enzymatic action. Two MW classes, one receptor-associated and one non-receptor associated, have been purified to homogeneity and their peptide composition is being studied. Functional studies reveal the larger MW forms derived from alloimmune cell populations; can induce selective and nonselective destruction of cells in vitro. Antibodies which inhibit the in vitro cell lytic ability of various human LT forms can block different classes of human lymphocyte cell killing reactions in vitro.

Isolation and identification of an α2 subclass lymphotoxin (LT) subunit from the high-molecular-weight (complex) human LT class

The high-molecular-weight (MW) complex class (MW = greater than or equal to 200,000) of lymphotoxin (LT) may be involved in the process of lymphocyte-mediated cytolysis. This LT class was produced by concanavalin A-stimulated human adenoid and tonsil lymphocytes in vitro and purified approximately 1000 fold by a scheme employing lectin affinity chromatography on concanavalin A-Sepharose, negative hydrophobic affinity chromatography on phenyl-Sepharose, and molecular sieving on Ultrogel AcA 44. A cell-lytic subunit, termed C-alpha toxin, was dissociated from this partially purified complex LT (Cx) preparation. The identification of C-alpha toxin as an alpha 2 LT form was established by its comigration with the alpha 2 LT form during native polyacrylamide gel electrophoresis (PAGE), molecular sieving, and isoelectricfocusing. In addition, C-alpha toxin was neutralized by antiserum raised against purified alpha 2 LT, and the latter was neutralized by antiserum raised against C-alpha toxin. Furthermore, preliminary evidence indicates that both alpha 2 LT and C-alpha toxin are composed of peptides with apparent MW of approximately 69,000. Additional data indicate the Cx LT form is composed of alpha 2 LT and other subunits.