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Surface antigen changes accompanying lymphocyte activation. Selective reactivity of certain anti-H-2 antisera with activated lymphocytes of unrelated H-2 haplotype.

It is here shown that some noncongenic anti-H-2 antisera produced by using normal spleen cells at the immunogen can contain cytotoxic antibody activity against activated, but not resting, lymphocytes of unrelated H-2 haplotype strains. Reactivity was noted against lymphocytes activated in vitro with T- or B-cell mitogens, or in vivo by antigenic stimulation. Analysis of the results indicated that the anti-activated lymphocyte activity was most likely related to production of anti-Ala-1 ('activated lymphocyte antigen') alloantibodies. This appears to occur as a result of donor lymphocyte activation during production of the anti-H-2 antisera, using Ala-1-incompatible donor and recipient combinations, where the donor cells are competent to recognize and react against host H-2 incompatibilities. These results confirm the finding that lymphocyte activation can be accompanied by the expression of an antigen that is either absent or weakly expressed on resting lymphocytes.

Animals

The biochemistry and in vitro activity of soluble factors of activated lymphocytes.

Activated lymphocytes release numerous products which are either synthesized de novo or in increased amounts; some of these products play a role in the regulation of the immune response and are designated as mediators of cellular immune reactions or lymphokines. The first lymphokine described was the macrophage migration inhibitory factor (MIF) which has been studied most extensively with regard to its chemical and biological properties. Using sensitive radiolabelling techniques and an antiserum against highly purified fractions of MIF we were able to identify several products of activated guinea pig lymphocytes with different molecular weights of 15.000, 30.000, 45.000, 60.000 which all had an isoelectric point of 5.2 and were all inhibitory to macrophage migration. It is suggested, that these molecules are oligomers of a common subunit of molecular weight 15.000. It was further shown, that molecules of the same physical-chemical and serological characteristics are produced by activated B-cells, L2C leukemia cells and growing fibroblasts, thus further substantiating earlier reports on the production of MIF by lymphoid and non-lymphoid cells. The described molecules were also shown not to contain determinants of the major histocompatibility complex and to be distinct from lymphotoxin, another lymphocyte activation product. It is concluded, that MIF is not a single molecule but rather a system of structurally related molecules. Their interaction with macrophages and possible relationships to macrophage activating factor is discussed.

Animals

Virus plaque assay: effective detection of virus plaque forming cells at the early stage of lymphocyte activation by mitogen and alloantigen.

Activated lymphocytes were detected quantitatively by virus plaque assay (VPA) during the course of lymphocyte cultures stimulated by mitogen or alloantigen. In Con A-stimulated cultures, the number of virus-plaque forming cells (V--PFC) was a more sensitive method of detecting the early stage of lymphocyte activation than [3H]-thymidine (3H-TdR) incorporation. This evidence was obtained by two methods of collecting cells of each stage. First, when Con A-activated lymphocytes were fractionated by velocity sedimentation at unit gravity to separate cell populations according to each cell stage, the ratio of the number of V-PFC to the radioactivity of incorporated [3H]-TdR was larger in the earlier stage of cell cycle than in the later stage. Second, when cultured lymphocytes were synchronized directly by addition of excess thymidine and colchicine, similar results were obtained. In primary mixed lymphocyte cultures, the generation of cytotoxic lymphocytes (CTL) was correlated better with the proliferative response than with V-PFC production. It was also found that both the incorporation of [3H]-TdR and the generation of CTL were abrogated by cytosine arabinoside (Ara-C) added to cultures up to one day before assay, whilst the generation of V-PFC was not so markedly affected by Ara-C. These findings suggest that V-PFC represent the number of precursor cells which require one or more generations to differentiate to CTL and not simply the number of effector lymphoyctes already exhibiting cytotoxicity.

Animals

A chemical approach to the mechanism of B-lymphocyte activation. I. The pure presentation of haptens does not activate B lymphocytes.

In an attempt to definitively determine whether the pure presentation of haptens (on repetitive, non-metabolizable carriers) to B lymphocytes triggers differentiation to antibody-producing cells, dinitrophenyl and trinitrophenyl groups have been covalently coupled to potentially inert carriers (polymethylmethacrylate, chlorinated polyethylene and cellulose). The resulting conjugates (with three different degrees of hapten substitution) are not immunogenic. It is therefore concluded that the pure presentation of haptens to B lymphocytes does not trigger antibody formation.

Acrylic Resins

The formation of stable E-rosettes by human T lymphocytes activated in mixed lymphocyte reactions.

The aim of the present study was to determine whether activation of human T-lymphocytes affects their interaction with sheep red blood cells (SRBC). Less than 3% of the E-rosettes formed by freshly isolated peripheral blood lymphocytes (PBL) and SRBC are stable and do not disintegrate after incubation at 37 degrees C. In contrast, about 30% of PBL kept in culture for 5 days in the presence of mitomycin C-treated allogeneic lymphocytes were found to form stable E-rosettes. Whereas no rosettes were formed by freshly isolated PBL incubated with human red blood cells at 24 degrees C, 15% of the cells recovered from mixed lymphocyte reactions (MLR) formed such rosettes. When responder PBL were maintained in culture in the absence of allogeneic stimuli the proportion of cells forming stable E-rosettes depended on the serum present in the medium. Less than 5% of the responder cells kept in medium containing human serum or in serum-free medium formed stable E-rosettes, whereas 18% of the cells maintained in medium containing fetal calf serum formed stable E-rosettes. The proportion of cells forming stable E-rosettes increased before any increase in DNA synthesis was detectable in MLR. Indeed, a high proportion of cells forming stable E-rosettes appeared in MLR taking place in serum-free medium, without any accompanying increase of DNA synthesis. Depletion of cells forming EAC'-rosettes from responder PBL increased the proportion of cells forming stable E-rosettes in MLR. Exposure of the cells recovered in MLR to specific anti-T sera inhibited the formation of both stable and regular E-rosettes. Exposure of the cells recovered in MLR to anti-Ig serum had no effect on the formation of regular rosettes. Anti-Ig serum strongly inhibited the formation of stable E-rosettes by cells grown in medium containing human serum, but had no effect on the formation of stable E-rossettes by cells grown in either serum-free medium or in serum containing fetal calf serum. It is concluded that activated human T lymphocytes are characterized by their capacity to form stable E-rosettes, resistant to incubation at 37 degrees C, and by their capacity to acquire an immunoglobulin coat, possibly by binding immunoglobulin molecules present in their environment.

Animals

The effect of membrane stabilizing agents on induction of the immune response. I. Effect of lymphocyte activation in mixed lymphocyte reactions.

The effect of three membrane-stabilizing agents, chlorpromazine, imipramine, and lidocain, on in vitro activation of cellular responses was studied using the one-way MLR. Cellular activation was found to be depressed by these drugs. However, the degree of suppression paralleled the decline of viable cells in the cultures. It is concluded that drug-induced inhibition of MLR was due solely to the toxicity of the compounds.

Animals

Effect of corticosteroids on lymphocyte activation.

Human peripheral blood lymphocytes were stimulated by concanavalin A, sodium periodate, and neuraminidase plus galactose oxidase. Response to mitogens was measured by the amount of tritiated thymidine incorporated as well as the percent of "giant sheep red blood cell rosettes" generated. The thymidine incorporation was diminished by the absence of monocytes or the presence of corticosteroids. The percent of giant rosettes generated was not influenced by either change. This finding suggested that considerable lymphocyte activation could still take place in the presence of corticosteroids. When subjects received 60 mg of prednisone, they developed lymphopenia 5 hr later. The circulating lymphocytes at that time responded less well to mitogen stimulation when measured by both thymidine incorporation and percent giant rosettes, suggesting a selective sequestration of mitogen-responsive lymphocytes outside the circulatory compartment.

Animals

Plasminogen activator production by human monocytes. I. Enhancement by activated lymphocytes and lymphocyte products.

Human monocytes, but not nylon wool column-nonadherent lymphocytes, produce plasminogen activator. The activity is found only in association with intact cells. Exposure of monocytes to activated lymphocytes or to lymphokine-rich supernatants enhances monocyte plasminogen activator production. The assay allows assessment of baseline and activated human monocyte function.

Antigens, Bacterial

Purification and properties of human lymphocyte activating factor (LAF).

Lymphocyte-activating factor (LAF) has been shown to be produced by LPS-stimulated human adherent cells (monocytes) and peripheral leukocytes, but many non-macrophage cell lines failed to produce LAF. Other macrophage activators including latex microspheres, antigen-antibody complexes, and barium sulfate induce the production of LAF. There is a delay of 6 hr before significant amounts of LAF activity appear in the supernatant medium and maximum activity is found after 12 to 24 hr. Chromatography of concentrated crude supernatant fractions containing LAF activity on Sephadex G-100 gave two peaks of activity (approximately 85,000 and 13,000 daltons). The latter constitutes the major activity and has been purified at least 500-fold with Sephadex G-100, anion exchange, and adsorption chromatography. Optimal stimulation with LAF induces mitosis in 10% of murine thymocytes. The purified activity is sensitive to chymotrypsin and is not affected by treatment with sodium periodate, sulfhydryl reagents, and phenylmethanesulfonylfluoride. The response of thymocytes to LAF decreases with age after 10 weeks and thymocytes obtained from animals injected with cortisone or tumor-bearing animals have an increased responsiveness to LAF.

Animals

Cyclic GMP and lectin-induced lymphocyte activation.

Purified human peripheral lymphocytes incubated with the mitogenic plant lectins phytohemagglutinin and concanavalin A were examined for alterations in intracellular cGMP and cAMP under a variety of experimental conditions and using multiple techniques for the isolation and purification of cGMP and cAMP before assay of the cyclic nucleotides by radioimmunoassay. In contrast to work reported by others, we have been unable to demonstrate consistent increases in cGMP under any of the experimental conditions used and with any of the various purification schemes. In these same experiments exogenous cGMP added to the lymphocytes could be measured, and the immunoreactive material was destroyed by cyclic nucleotide phosphodiesterase, indicating that our inability to measure increases in cGMP was not caused by our inability to measure cGMP. Under identical experimental conditions, small but consistent and statistically significant increases in cAMP were noted. In addition, other parameters of lymphocyte activation, 45Ca uptake (an early parameter), and incorporation of 3H thymidine into DNA were unimpaired. These data call to question the concept of cGMP as the second messenger in lectin-stimulated human peripheral lymphocytes.

2',3'-Cyclic-Nucleotide Phosphodiesterases