Membrane alteration responsible for the induction of gamma interferon.

Induction of gamma interferon in human lymphoid cells cultures appears to be dependent upon specific membrane-mediated events and calcium flux. Since blastic response had been observed after enzymic oxidation of membrane-bound galactose residues, we used this system to study the nature of membrane alterations responsible for the activation of interferon induction. The results of these experiments suggest that a membrane oxidation is essential for interferon induction and depletion of calcium abolishes interferon production. In addition, we have shown that interferon induction by concanavalin A, phytohemagglutinin, and staphylococcal enterotoxin A, but not by galactose oxidase is prevented by cleavage of N-acetylneuraminic acid residues. Thus, interferon induction in human lymphoid cell cultures by galactose oxidase, concanavalin A, phytohemagglutinin, staphylococcal enterotoxin A, and NaIO4 appears to reside in terminal oligosaccharides of the cell membrane. How this specific membrane event relates to the derepression of the interferon locus is being actively pursued.

Human immune interferon: induction in lymphoid cells by a calcium ionophore.

Human lymphocyte cultures produced interferon after treatment with the calcium ionophore A23187, but not after treatment with potassium ionophore nonactin. Interferon production was detectable between 3 and 6 h after ionophore treatment and reached the maximum level between 12 and 24 h. Ionophore-induced interferon appeared to be immune interferon on the basis of acid lability, lack of neutralization by antibody to leukocyte interferon, slow kinetics of activation of the antiviral state, and molecular characterization by isoelectrofocusing.

Observations on the level of cyclic nucleotides in three population of human lymphocytes in culture.

The level of cyclic nucleotides in three populations of cultured human lymphocytes were studied. An early conspicuous elevation of c-GMP level and a reciprocal relationship between c-AMP and c-GMP fluctuations were demonstrated in T cells from normal donors. Null cells from patients with ALL showed a constantly low level of c-AMP, while c-GMP fluctuated in apparent relationship with cell doubling time. Persistently low levels of c-AMP and persistently high level of c-GMP were found in B cells from patients with CLL. Possible significance of these findings is discussed.

Enzymatic induction of interferon production by galactose oxidase treatment of human lymphoid cells.

Human lymphocyte cultures produced large amounts of interferon after treatment with the enzyme galactose oxidase. Interferon production was detectable as early as 3 h after enzymatic treatment and reached a level of about 10(4) reference units 20 to 24 h later. Galactose oxidase-induced interferon appeared to be immune interferon on the basis of acid lability, lack of neutralization by antibody to leukocyte interferon, and slow kinetics of activation of the cellular antiviral state. Interferon production was inhibited to the same extent (99%) by pretreatment of the cells with beta-galactosidase or with neuraminidase followed by beta-galactosidase, suggesting that the critical event for activation of interferon production is the oxidation of exposed galactose residues on lymphocyte membrane.

Cell interaction during lymphocyte activation.

Adherent cells were separated from stimulated lymphocytes by a layer of agarose to determine the requirement for lymphocyte interaction with adherent cells. The results demonstrated that the presence of plastic adherent cells was obligatory for oxidative activation, but contact of plastic adherent cells with oxidized nonadherent lymphocytes was not. A similar relationship was found to exist between adherent and nonadherent populations during PHA and Con A stimulation. In contrast, the responses of PWM- and ZnCl2-stimulated lymphocytes were not augmented by the presence of adherent cells unless the two populations were in contact.

Increased purine nucleotides in adenosine deaminase-deficient lymphocytes.

It was found that ATP and cyclic AMP were greatly increased in human blood lymphocytes which were deficient in ADA. Certain other purine and pyrimidine nucleotides were elevated but to a lesser degree. Energy production in these cells may be inhibited by the increase in nucleotides since the ATP:ADP ratio was significantly below normal. Thus it appears that the immunologic deficiency in human ADA deficiency is related to increased nucleotide concentrations in the lymphocytes.

Stimulation of human peripheral blood lymphocytes by a sodium periodate (NaIO4) induced growth factor.

Sodium periodate treated lymphocytes engage in cell to cell interaction and also yield a soluble growth factor that stimulates DNA synthesis when added to naive autologous cells. Adherent cells enhance the factor mediated activity. Sodium periodate stimulated lymphocytes, treated with mitomycin C, produced no growth factor activity. Partial characterization of the factor indicates that it is non-dialyzable, resistant to ribonuclease, and sensitive to heat, trypsin, and papain.

Abnormal ultrastructural changes in sodium periodate-stimulated lymphocytes from patients with chronic lymphocytic leukemia.

Lymphocytoid and plasmacytoid blasts have been identified in both normal and chronic lymphocytic leukemia lymphocyte cultures exposed to NaIO4. However, the appearance of ultrastructurally abnormal blasts in chronic lymphocytic leukemia cultures suggests that NaIO4 also stimulates the transformation of an abnormal subpopulation of lymphocytes. Development of invaginated nuclei produced morphological features similar to nuclear blebs, a cellular abnormally described in blood cells from other cancers. Furthermore, the consistent localization of nuclear invagination only to portions of nuclei adjacent to developing cytoplasmic microtublar complexes suggests a role for microtubules in the transformation process. The absence of these unusual blasts in cultures stimulated with other mitogens may indicate that these NaIO4-sensitive cells are not responsive to the more commonly used plant lectins.

Serum factor: the inhibition of phytohemagglutinin-induced blastogenesis of chronic lymphocytic leukemia lymphocytes.

A serum factor (SF), isolated from acidified sera of normal donors, inhibited phytohemagglutinin (PHA) stimulation of lymphocytes isolated from patients with chronic lymphocytic leukemia (CLL),but had no effect on those from healthy donors. An SF isolated by an identical procedure from the sera of patients with CLL had essentially no inhibitory effect on DNA synthesis in normal or leukemic lymphocytes. The SF was isolated from sera or plasma by ammonium sulfate precipitation, acidification to pH 1.5, and chromatography on DEAE-cellulose. The SF was heat labile and sensitive to digestion by trypsin. Maximum inhibition was obtained when the factor was added within 24 hours after the cells were stimulated with PHA.

Cyclic adenosine 3':5'-monophosphate levels and activities of related enzymes in normal and leukemic lymphocytes.

The role of cyclic adenosine 3':5'-monophosphate (cyclic 3':5'-AMP) in the regulation of cell division in lymphocytes obtained from healthy donors and from patients with chronic lymphocytic leukemia (CLL) was investigated by determining the levels of cyclic 3':5'-AMP and glycogen and also the activities of several enzymes that are closely associated with the metabolism of these cellular components. Intracellular levels of cyclic 3':5'-AMP were measured in normal and CLL lymphocytes in nondividing, dividing, and quiescent [after phytohemagglutinin (PHA) addition]states. In normal lymphocytes the levels of cyclic 3':5'-AMP fluctuated throughout the cell cycle after PHA addition, whereas in CLL lymphocytes the levels were approximately 3-fold lower than in normal cells and remained relatively constant before, during, and after mitogenic stimulation. Normal cells contained approximately 3-fold lower levels of glycogen than CLL cells, whereas glycogen phosphorylase activities were increased 2- to 4-fold above those in nondividing cells in normal but not in CLL lymphocytes after stimulation with PHA. Furthermore, cyclic 3':5'-AMP phosphodiesterase activities were higher in CLL lymphocytes than in normal ones. Collectively, these studies demonstrated that (a) the intracellular levels of cyclic 3':5'-AMP differ in these two cell types; (b) the levels of cyclic 3':5'-AMP and glycogen qualitatively correlate with the activities of the enzymes that are related to these components; and (c) an inverse relationship between the levels of cyclic 3':5'-AMP and cell growth exists in mitogen-stimulated lymphocytes from healthy donors but not from patients with CLL. These biochemical differences are presumed to exist between normal and "leukemic" lymphocytes, but alternatively they may reflect normal populations of immunologically distinct lymphocytes.