Lymphokine properties of a lymphoid cultured cell supernatant fraction active in promoting tumor regression

doi:10.1016/0090-1229(76)90148-3

Clinical Immunology and Immunopathology

Volume 5, Issue 1, January 1976, Pages 48-59

Clinical Immunology ...

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Abstract

In the present study supernatant culture medium from a cultured human lymphoid cell line, which was shown to have inflammation-inducing and tumor-regressing properties in man, was tested for lymphokine properties. Skin reactive factor, lymphotoxin, macrophage activation factor, and chemotactic factor were assayed and found to be present in the crude, active fraction that was confined to a size of 10⁴–10⁵ daltons and was active in producing clinical tumor lesion regression. These results suggest that the in vivo antitumor effect is related to lymphokines present in the active preparation.

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Cellular origin of human lymphotoxin and its purification
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The ability of various subsets of human mononuclear cells to produce human lymphotoxin (LT) was examined. Peripheral blood mononuclear cells were separated into OKT4⁺, OKT8⁺, and Leu-lla⁺ subpopulations by flow cytometry. Both OKT4⁺ and OKT8⁺ cells produced LT upon phytohemagglutinin (PHA) stimulation, but the OKT4⁺ T cells were the major source of LT. In contrast, Leu-lla⁺ cells failed to produce LT. The LT production and the proliferative response to PHA, of OKT4⁺ and OKT8⁺ cells, were inhibited by prostaglandin E₂ and histamine. The LT, derived from PHA-stimulated peripheral blood lymphocytes, was purified by a procedure involving Blue-agarose, Con A-Sepharose chromatography, preparative gradient polyacrylamide gel electrophoresis and preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The LT activity was recovered from SDS gel with major activity peak in the M_rmr 76,000 region and the minor activity peak in the M_rmr 24,000 region. The LT derived from 1788 lymphoblastoid cell line also showed heterogeneity on SDS gel. The activity was recovered from two peaks in the region of M_rmr 70,000 and 20,000.
Utilization of purified human monocytes in the agarose droplet assay for measuring migration inhibitory factors
1983, Journal of Immunological Methods
Human monocytes, highly purified by counter-current elutriation, are excellent indicator cells for evaluation of human migration inhibitory factors (MIFs). We have adapted the agarose droplet MIF assay initially developed for guinea pig peritoneal exudate cells to utilize human monocytes. The experimental variables have been evaluated and standardized to make this assay a quantitative and sensitive method for measuring MIF activity. The assay can be performed serum-free in RPMI 1640 medium without protein or hormone additives, thereby increasing the sensitivity and eliminating potential masking of MIF effects by serum components. Cryopreserved monocytes also performed well in this assay, migrating approximately the same distance per unit time and showing migration inhibition in response to inhibitory factors. This assay provides a powerful tool in evaluating MIF-like activities of various lymphokines and factors, and could be used to monitor the activity of fractions produced during the physicochemical separation of MIFs from lymphokine-containing supernatants.
Lymphokine-stimulated macrophage phagocytosis of fluorescent microspheres: a rapid new assay
1983, Journal of Immunological Methods
A highly sensitive and rapid in vitro macrophage phagocytosis assay is described for screening of lymphokine preparations with macrophage activating properties. Non-induced mouse peritoneal macrophages were incubated on glass slides for 60 min in the presence of fluorescent 2 μm latex beads and partially purified lymphokine fractions or media control. Lymphokine samples were prepared from culture supernatants of the B lymphoblastoid cell line RPMI 1788 by high performance liquid chromatography of a soluble trichloroacetic acid extract of concentrated culture supernatant. Phagocytosis was measured by direct counts of the percent phagocytic cells and the number of intracellular beads per 100 cells as seen by fluorescence microscopy. Phagocytic uptake in the presence as little as 0.1 ng of active lymphokine could readily be determined qualitatively by correlation plots and quantitatively by appropriate statistical programs for data processing by computer. This technique provides a rapid, reproducible, screening assay for biological activity of macrophage activating lymphokines and other compounds affecting macrophage phagocytosis.
Biological and physicochemical characterization of keyhole limpet hemocyanin-lnduced guinea pig lymphotoxin
1982, Cellular Immunology
Biological and physicochemical properties of keyhole limpet hemocyanin (KLH)-induced guinea pig lymphotoxin (LT) were compared to those of LT induced by another antigen, ovalbumin, or by the mitogen, phytohemagglutinin (PHA). Mitogen- and antigen-induced LT had similar colony inhibitory activities toward guinea pig and rat tumorigenic cells but none toward nontumorigenic cells. Conversely, human nontumorigenic but not tumorigenic cells were inhibited by the three LT preparations. The molecular weight of each LT was 45,000 daltons on gel filtration chromatography but sucrose density gradient ultracentrifugation indicated greater heterogeneity. The KLH-, ovalbumin-, and PHA-induced LTs were resolved into multiple peaks upon preparative column isoelectric focusing (IEF) with IpH's in the range of 4.5 to 5.3. Analytic IEF of the KLH-induced LT revealed the presence of three distinct biologically active peaks with IpH at 4.77, 5.02, and 5.20. LT activity was stabilized during preparative IEF by inclusion of 0.1% polyethylene glycol (4000 molecular weight) in the ampholine gradient. This resulted in 90% recovery of LT activity with a concomitant removal of 96% of the KLH used as the antigenic stimulus for LT production. Fractionation of LT activity during diafiltration and IEF when exogenous proteins were included as protective agents altered the number and the isoelectric characteristics of the peaks obtained suggesting that LT was being bound by the added protein. Colony inhibitory (cytostatic) and ³H-release (cytolytic) LT activities copurified during fractionation. Thus, this study demonstrated that high yields (approximately 50% of the starting material) of LT, free of antigen or exogenous serum proteins, can be obtained by a combination of diafiltration and IEF performed in the presence of polyethylene glycol.
Statistically designed assay of migration-inhibitory activity in lymphokine preparations
1981, Immunopharmacology
The migration inhibition test in agarose as described by Clausen (Acta Allergol 26:56, 1971) was modified into a statistically designed assay comprising a laboratory standard in each assay, and using buffy coat cells or peripheral polymorphonuclear leukocytes from pigs as target cells. The precision of the assay was improved by combining cells from several pigs. Fifty-5000 microliters of lymphokine-containing test fluid could be used in the assay provided that this fluid contains the essential nutrients. In our hands, the capillary test was unsuitable for a quantitative assessment of migration inhibition activity.
Biological characterization of guinea pig lymphotoxin production
1979, Immunopharmacology
The generation of serum-free lymphotoxin by antigen-stimulated ovalbumin immune strain 2/N guinea pig peritoneal leukocytes in vitro is dependent upon a number of common variables in cell culture. Lymphotoxin production varies directly with antigen concentration, duration of antigen exposure and leukocyte culture, leukocyte density, and medium volume. The maximum concentration of lymphotoxin generated in the medium has a limiting value that is independent of antigen concentration, leukocyte density, and duration of antigen exposure or leukocyte culture, suggesting feedback control of lymphokine production. Optimal conditions for the reproducible maximum generation of guinea pig lymphotoxin were 10⁷ oil-induced peritoneal leukocytes from immune animals cultured for 12–24 hr in 20 ml RPMI 1640 medium containing 100 μg ovalbumin/ml throughout the period of culture.

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^☆: These studies were supported in part by funds from the Cancer Research Institute, Inc., New York; Hematology Research Fund, Mercy Catholic Hospital; The University of Texas; and NIH DHEW Grant No. 5S01 RR05427(12).

²: At the time these studies were performed, Steven Parmett, formerly an undergraduate student at Harvard College, and Dennis Dobkin, formerly an undergraduate student at the University of Rochester, were NSF Summer Fellows at Roswell Park Memorial Institute, Buffalo, N.Y. Present addresses: S. Parmett, Harvard Medical School, Boston, Mass., and D. Dobkin, SUNY Medical School, Syracuse, N.Y.

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Lymphokine properties of a lymphoid cultured cell supernatant fraction active in promoting tumor regression☆

Abstract

Clin. Immunol. Immunopathol.

Blood

J. Med. (Cincinnati)

Res. Commun. Chem. Pathol. Pharmacol.

Amer. J. Pathol.

J. Immunol.

J. Exp. Med.

J. Immunol.

J. Exp. Med.

J. Immunol.

Clin. Immunol. Immunopathol.