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PubMed1978

Characterization of lymphocyte-activating factor (LAF) produced by a macrophage cell line, P388D1. II. Biochemical characterization of LAF induced by activated T cells and LPS.

Unstimulated P388D1 cells, as well as P388D1 cells stimulated with PHA-activated guinea pig T lymphocytes or LPS, produced a lymphocyte activating factor (LAF). In order to have a chemical basis for comparing this LAF with the LAF produced by normal macrophages, we have analyzed several biochemical characteristics of the P388D1-derived LAF. Sephadex G-75 chromatography of concentrated LAF-containing supernatants from cultures of unstimulated and T cell stimulated P388D1 cells demonstrated that the cell line LAF had a m.w. of approximately 16,000. On DEAE cellulose, the T cell-induced LAF fractionated into at least three major peaks and one minor peak. By using hydroxylapatite chromatography, two of the major peaks of LAF activity were separated from residual contaminating Lowry positive material. LPS-stimulated P388D1 also produced LAF with a m.w. of 16,000. However, the LPS-induced LAF appeared to lack one of the DEAE peaks of LAF activity observed with the T cell-derived LAF. In contrast to LPS, T cells may induce the synthesis and/or release of an additional LAF component or enzymatically modify one or more of the LAF species that are produced in response to both stimulants. Based on the results of chemical characterization studies, the P388D1-derived LAF appears to be similar in size and charge to the lymphocyte activating factor produced by normal macrophages.

Animals↗
PubMed1978

Biochemical relationship of thymocyte mitogenic factor and factors enhancing humoral and cell-mediated immune responses.

Supernatants from concanavalin A-induced mouse spleen cells contain soluble factors which: 1) augment the anti-sheep erythrocyte hemolytic plaque-forming cell response of nude mouse spleen cells, 2) facilitate the generation of antigen-specific cytotoxic cells by alloantigen-stimulated thymocytes, and 3) enhance the mitogenic response of thymocytes in the presence or absence of phytohemagglutinin. The uninduced control spleen cell supernatants contained variable but significantly less biologic activity within the difference assay systems. The principle enhancing activity (apparent m.w. 35,000 to 38,000) in each of the assay systems was found to be inseparable after a multiple step purification scheme involving ammonium sulfate precipitation, two-cycle gel filtration, hydroxylapatite chromatography, and hydrophobic chromatography on Phenyl Sepharose. These results indicate that either a single factor (designated thymocyte mitogenic factor) augments both humoral and cell-mediated immune responses or, alternatively, the different biologic activities are mediated by different factors that share a common core structure.

Ammonium Sulfate↗
PubMed1978

Lymphocyte-activating factor. I. Generation and physicochemical characterization.

Lipopolysaccharide-activated murine peritoneal macrophages elaborate lymphocyte-activating factor (LAF) which is mitogenic for murine thymocytes. A method of LAF production is presented that permits the generation of a relatively homogeneous molecular species. LAF has an isoelectric point of 4.8 (range 4.7-4.9). The m.w. was determined by using several physical techniques. The apparent sedimentation coefficient (S20,w) was determined to be 2.0S by sucrose gradient ultracentrifugation. The Stokes (molecular) radius was determined by gel filtration on Sephadex G-75 to be 22 A (range 21.5 to 22.5); the calculated diffusion coefficient (D20,w) was 9.7 X 10(-7) cm2/sec (range 9.5 X 10(-7) to 9.9 X 10(-7). The buoyant density of LAF is 1.30 g/cm3 (range 1.27 to 1.33) as determined by CsCl isopycnic ultracentrifugation; the partial specific volume was estimated to be 0.72 (range 0.70 to 0.74). From these data, the m.w. was calculated to be 18,000 daltons (range 16,400 to 19,600) with the Svedberg equation. The frictional ratio was calculated to be 1.25.

Animals↗
PubMed1979

Purification and biochemical characterization of human lymphocyte mitogenic factor (LMF).

Supernatants of human T lymphocytes stimulated by TT antigen release two factors that induce mitogenesis in autologous and allogeneic B lymphocytes. These factors are precipitated by 60% ammonium sulfate and 50% ethanol, and are both destroyed by heating to 70 degrees C for 5 min. By equilibrium ultracentrifugation there was a peak of mitogenic activity in the fraction with a specific gravity of 1.3147 corresponding to a partial specific volume of 0.761. After ultrafiltration through an Amicon XM50 membrane, the concentrate was chromatographed on a Sephadex G-200 column. Mitogenic activity was found only in the post-albumin fraction. When the post-albumin fraction was run on an isoelectrofocusing column, two distinct mitogenic factors were identified. The major peak of mitogenic activity (LMF) had a pI of 6.68 +/- 0.05 and the minor peak (MMF) had a pI OF 7.27 +/- 0.05. Amino acid analysis of LMF identified it as a protein and PAGE showed that LMF probably was a tetramer with a m.w. of 80,000.

Amino Acids↗
PubMed1979

Physicochemical characterization of lymphocyte-activating factor (LAF).

The LAF produced by the mouse macrophage cell line, P388D1, is a single polypeptide chain of m.w. 12,000 to 16,000 daltons. Native LAF was destroyed by Streptomyces griseus protease, but not by trypsin, chymotrypsin, and papain, although in the presence of 8 M urea, papain completely destroyed LAF activity. LAF did not bind to concanavalin A-Sepharose, suggesting that LAF does not contain significant amounts of mannosyl or glycosyl residues. Since LAF activity was not inactivated by a treatment of reduction and alkylation the active conformation of LAF does not appear to be dependent on disulfide linkages. LAF was not irreversibly denatured by 8 M urea or 0.1 to 0.5% SDS. On SDS-polyacrylamide gels, the m.w. of LAF was 12,000 daltons, as compared to a value of 16,000 daltons, as determined by gel filtration. The isoelectric point of LAF was 5.0 to 5.4 as determined on 7.5% acrylamide gels (pH 3 to 10). On the basis of these results it appears that the P388D1 cell line-derived LAF is a relatively stable molecule that shares several physicochemical properties with normal human and mouse macrophage-derived LAF.

Alkylation↗
PubMed1979

Isolation from lactalbumin hydrolysate of a high molecular weight mitogenic factor.

A new mitogenic factor has been isolated from tissue culture grade lactalbumin hydrolysate. Incubation of postconfluent Swiss 3T3 cells in serum-free medium containing lactalbumin hydrolysate resulted in enhanced synthesis and release of plasminogen activator. Sephadex G-100 gel filtration of concentrated, dialyzed lactalbumin hydrolysate revealed two fractions, LH-FI and LH-FII. LH-FI elutes at the void volume, indicative of high molecular weight, and contains all plasminogen activator stimulatory activity of the original lactalbumin hydrolysate, whereas LH-FII has no activity. In addition, LH-FI also induces DNA synthesis in Swiss 3T3 cells in a dose-dependent fashion, 1 to 2 microgram/ml being equivalent to 10% fetal calf serum. Again, LH-FII is without effect. Induction of DNA synthesis in LH-FI or serum-stimulated quiescent sparse cells followed essentially identical kinetics at least through the first 20 h. Furthermore, LH-FI also enhances 3T3 cell growth. Preliminary results of Sepharose 4B filtration of LH-FI reveal the presence of five subfractions each with plasminogen activator stimulatory as well as mitogenic activity.

Animals↗
PubMed1979

[Characteristics of the mitogenic activity of different staphylococcal strains].

The mitogens of human peripheral lymphocytes were detected in the metabolic products of 59 out of 71 S. aureus strains. The preparations of S. epidermidis were inactive. When stimulated with filtrates of various S. aureus strains, 72-hour lymphocyte cultures were found to have 0--46% of blasts. Two-year observation of a strain showed its stable mitogenic characteristics. The mitogenic properties of the preparations did not correlate with their coagulase, alpha-toxic, dermonecrotoxic, cytotoxic, enterotoxigenic, neutrophil-stimulating activity and the quantitative content of A protein.

Cells, Cultured↗
PubMed1977

Subpopulations of lymphocytes to produce various lymphokines. I. Function of subpopulations separated by velocity sedimentation.

Identification of a subpopulation of lymphocytes producing lymphokines was attempted by fractionating the lymph node cells from guinea pigs immune to DNP-BSA by velocity sedimentation at 1 x G. Each of six fractions obtained by this procedure was cultured with or without the presence of antigen, and the culture supernatants that were separated 24 hr later were assayed for various lymphokine activities. Most of the lymphokines, including migration inhibition factor, chemotactic factor for neutrophils, mitogenic factor, and lymphotoxin were generated by the first two fractions of lymphocytes, which represented the largest, most rapidly sedimenting cells. Although th procedure of cell separation does not depend on cell surface properties, the larger cells contained more cells with T cell surface markers and the smalller contained more cells with B cell surface markers. Proliferative response of those lymphocytes measured by 3H-thymidine uptake, however, has shown that the largest two subpopulations responded poorly either to specific antigens or to mitogens (PHA and LPS), and rather that the medium size cells responded most strongly to the both stimulants. These results indicated that the production of some lymphokines confined to certain subpopulations of lymphocytes which are large in size. Further, these cells are readily separable from the medium sized cells that respond strongly to antigenic and mitogenic stimuli with mitogenic responses.

Animals↗
PubMed1978

Lymphocyte mitogenic factor in sera from patients with falciparum malaria.

To test for the presence of a lymphocyte mitogenic factor in malaria, sera were obtained from 10 patients with malaria (9 with falciparum and one with vivax), and 10 noninfected controls. The sera from the malarial patients caused an increased blastogenesis in mouse splenic lymphocyte cultures and inhibited hemagglutination between lipid-A-coated erythrocytes and lipid-A antibodies. None of the sera were positive using the limulus amebocyte lysate test. These results could be interpreted to demonstrate that patients with falciparum malaria have a circulating mitogen which cross-reacts with endotoxin. However, alternate explanations must be considered, including an hypothesis that antiglobulins and/or immune complexes in the sera of malarious patients both caused the blastogenesis of mouse spleen cells and inhibited hemagglutination to lipid-A antibodies.

Adult↗