The regulation of lymphotoxin release from stimulated human lymphocyte cultures: The requirement for continual mitogen stimulation

Daynes, Raymond A.; Granger, Gale A.

doi:10.1016/0008-8749(74)90077-x

Cited by 22 publications

(6 citation statements)

References 25 publications

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“…Previous and present results substantiate that a polyclonal lectin signal triggers release, within 5 hr, of cell-lytic molecules accumulated in the intracellular pools of preactivated human iymphocytes (1)(2)(3). Extensive studies reveal the LT forms in these supernatants are heterogeneous in their size (14,15), cell-lytic capacity (3,11,16,17), and their ability to bind to different cell types in vitro (3,6,17).…”

Section: Discussionsupporting

confidence: 83%

Inhibition of human NK-induced cell lysis and soluble cell-lytic molecules with anti-human LT antisera and various saccharides

Weitzen

Innins

Yamamoto

et al. 1983

Cellular Immunology

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The present study examines and compares the cytolysis of K-562 and MOLT-4 cells mediated by human natural killer (NK) cells from fresh peripheral blood and lymphotoxins (LT) derived from human lymphoid cell populations after lectin stimulation in vitro. Lymphotoxins were obtained from 5-hr concanavalin A (Con A)-restimulated human peripheral blood lymphocytes (PBL) which were precultured for 5 days in medium and fetal calf serum or with allogeneic human B-lymphoid cell lines. Two classes of probes were employed in both direct (cell) and indirect (supernatant) induced target-cell lysis: (a) various saccharides and (b) antibodies reactive with human LT forms. Two sugars, N-acetylglucosamine and a-methylmannoside, were able to inhibit direct cell lysis of both MOLT-4 and K-562 target cells. However, saccharide inhibition was distinct for each type of target even when effector cells were obtained from the same donor. These same saccharides were also able to inhibit 20-30% of the total LT activity in a supematant for L-929 cells and 50-90% of the lytic activity on MOLT-4 cells. Anti-human F(ab')t (IgG) and rabbit anti-q LT sera blocked direct cell lysis of MOLT-4 and K-562 targets in 50% of the experiments. The anti-a* LT serum only recognizes a portion of the LT forms in these supernatants. These results reveal that, while both direct and indirect cell lysis are complex phenomena, they may both occur in some cases by a common mechanism(s).

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Section: Discussionsupporting

confidence: 83%

Inhibition of human NK-induced cell lysis and soluble cell-lytic molecules with anti-human LT antisera and various saccharides

Weitzen

Innins

Yamamoto

et al. 1983

Cellular Immunology

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“…The cultures were established under two basic experimental protocols: (l)"primary" activation, effected by adding Con-A up to 20 µg/ml to nonactivated cells, and (2) "secondary" stimulation of previously activated cells, which were temporarily deactivated. The latter procedure has been described in detail previously (3,4) . Briefly, after a 48-hr activation by incubation with Con-A at 20 µg/ml, the cells were washed free of Con-A by the addition of phosphate buffered saline (PBS, 0.15 M NaCl, 0.01 M PO, buffer, pH 7.2) containing 5 x 10-2 M a-methyl-mannoside (MAM, Sigma), a competitive inhibitor of Con-A.…”

Section: Methodsmentioning

confidence: 99%

The Effect of Agents Which Modulate Levels of the Cyclic Nucleotides on Human Lymphotoxin Secretion and Activity in Vitro

Prieur¹,

Granger²

1975

Transplantation

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The ability of concanavalin-A (Con-A) to activate lymphocytes to secrete human lymphotoxin (LT) was tested in the presence of agents known to modify the intracellular levels of cyclic nucleotides (cAMP and cGMP). Lymphocytes were treated with these agents at different stages of activation: (1) during the first encounter with mitogens, and (2) after they had been fully activated and were restimulated. Two agents, Dibutyryl cAMP and theophylline, dramatically inhibit the amount of LT secreted during both "primary" and "secondary" activation by Con-A. In contrast, DL-isoproterenol had a weak effect during primary activation, but greatly reduced the level of LT secreted during secondary activation. Agents which affect the intracellular level of cGMP were also tested. Imidazole had no effect on LT secretion by either primary or secondary Con-A activated cells. In contrast, carbamyl choline greatly reduced LT secretion to a level comparable to Dibutyryl cAMP and theophylline. All agents tested protected, to some degree, the sensitive a-L cell against LT-induced destruction in vitro. Agents which affect the levels of cyclic nucleotides affect both the effectiveness of the aggressor cell and the sensitivity of the target cells in vitro.Cyclic nucleotides have been shown to have an important role in the regulation of cellular biosynthesis and metabolism (19) in inflammation and immunity (1). They appear to be important in both immediate and delayed hypersensitivity reactions. Production and/or secretion of antibodies by B lymphocytes is reduced when intracellular levels of cyclic adenosine 3',5'-monophosphate are raised (16,23). Destruction of cells by immune lymphocytes activated by contact with target cell antigens, and in vitro measure of cell-mediated immunity, is suppressed when the effector lymphocytes are treated with pharmacologic drugs which increase the adenosine 3',5'-monophosphate (cAMP) level (14,21,22) . On the other

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“…Throughout the course of these experiments it had been noted that some of the cell cultures when cut back to a low density experienced a marked lag phase before exponential growth (Figs. 3,8,9,11,and 12). It has now been recognized that when cells remain in the stationary phase of growth for 24 or more hours before being resuspended a t a low cell density in fresh medium they undergo a lag phase before exponential growth occurs.…”

Section: 53mentioning

confidence: 99%

An evaluation of suspension culture systems for the growth of murine lymphoblastoid lines expressing TL and Thy‐1 alloantigens

Zwerner

Runyan

Cox

et al. 1975

Biotech & Bioengineering

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4s a prelude to our studies on T L and Thy-1 differentiation alloantigens, three rnurine Iymphoblastoid cell lines were examined for expression of these components. Optimal conditions for their m i i s culture were also determined. Several suspension culture systems were evaliiattd: a) 50 ml through 500 ml Wheaton and Bellco spinner flasks as well as 1, 4, and 8 liter Whcaton flasks modified for semicontinuous culture conditions, b) 3 lit.er Chemapec Vibrofermentor, and (1) 14 liter New Brunswick fermcntor. Utilizing these typcs of vessels the opt.iinal culture conditions were evaluat,ed as to Lhe effect. of: 1 ) pH, 2) initial concentration of cell inoculum, 3) t.ypes of media, and 4) methods of gmsing and gas mixtures on t,he rat.e of growth and alloant.igen expression. This study dcnionstrateti that cells could be cultured on a sernicont.inuous b:tsis up t.o densities of 2-4 X 106 cells/ml if a vessel of appropriate dimensions was utilized, t.he appropriate medium select.ed, and the p H controlled by CO, and air overlay. Once these paranieters were est,ablished the growth of a given cell line was highly reproducible. Under optimal culture conditions the expression of Thy-1 was maximum while t.he cells were in the exponential stage of growth and reduced during the lag and stationary p h u e s of growth. The expression of T L did not vary as significantly during the various stages of growth. One cell line grown in medium supplemented with 10c/r horse serum expressed less Thy-I than those grown in medium cont.aining 10%. fetal calf serum. The factors affecting cell growth and alloantigen expression have been considered in the design of a large-scale suspension cult.ure facility for cult.uring 1000 liters of cells per week.

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The regulation of lymphotoxin release from stimulated human lymphocyte cultures: The requirement for continual mitogen stimulation

Cited by 22 publications

References 25 publications

Inhibition of human NK-induced cell lysis and soluble cell-lytic molecules with anti-human LT antisera and various saccharides

Inhibition of human NK-induced cell lysis and soluble cell-lytic molecules with anti-human LT antisera and various saccharides

The Effect of Agents Which Modulate Levels of the Cyclic Nucleotides on Human Lymphotoxin Secretion and Activity in Vitro

An evaluation of suspension culture systems for the growth of murine lymphoblastoid lines expressing TL and Thy‐1 alloantigens

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