In vitro production of immune interferon (IFN gamma) by murine spleen cells when different sensitizing antigens are used in vivo and in vitro

OK-432, a killed preparation of Streptococcus pyogenes, as well as bacillus Calmette-Guérin (BCG) and Corynebacterium parvum are all known to induce immune interferon (IFN gamma) in mice. To examine the mechanisms of IFN gamma induction by OK-432, DDI mice were sensitized with various doses of OK-432, either by a single injection of a 1-mg dose or repeated injections of 0.1-mg doses given intraperitoneally. Spleen cells removed from the mice 7-9 days after the last injection produced high-titered IFN gamma (600-800 IU/ml) in vitro in the presence of 5 micrograms/ml of OK-432. In the absence of OK-432, however, in vitro IFN gamma production of sensitized spleen cells was quite limited. Moreover, when inducers of different antigenic entities such as serologically unrelated Streptococcus faecalis, Listeria monocytogenes, or Con A were added in vitro, instead of OK-432, to the OK-432-sensitized spleen cells, high-titered IFN gamma production also occurred. This may indicate that the signal required by T cells to produce IFN gamma in vitro need not necessarily be the same as that required to sensitize mouse macrophage in vivo. Once sensitized with OK-432, mice spleen cells continued to produce high-titered IFN gamma for more than 3 but less than 5 weeks.     

Interleukin 2 regulation of mitogen induction of immune interferon (IFN gamma) in spleen cells and thymocytes

Interleukin 2 (IL 2) or T-cell growth factor induced the production of immune interferon (IFN_γ) in C57B1/6 mouse spleen cell cultures, and enhanced mitogen-induced IFN_γ production in both spleen cells and thymocytes. Staphylococcal enterotoxin A, but not phytohemagglutinin P (PHA-P), induced IFN_γ production in thymocytes. IL 2 enhanced this production by almost 12-fold, while having no effect on the negative response to PHA-P. The IFN activity was shown to be IFN_γ by neutralization with specific antiserum. A strict correlation between IL 2 induction or enhancement of IFN_γ production and cell proliferation was not observed, probably indicating that non-IFN-producing cells also proliferated in the presence of IL 2. The data indicate that IL 2 can both induce IFN_γ and modulate mitogen induction of IFN_γ.     

Characterization of immune type interferon (IFN gamma)-induced cytoplasmic protein kinase activity in mouse L cells

Mouse immune interferon (IFNγ) induced double-stranded RNA-independent protein kinase activity in the cytoplasmic fraction of mouse L cells as measured against a histone substrate. Chromatographic purification separated the activity into three distinct kinases of molecular weights of approximately 100K (kinase I), 70K (kinase II), and 70K (kinase III). Partially purified IFNγ was as effective as crude in inducing protein kinase activity. Induction was blocked by treatment of IFNγ with specific anti-IFNγ antibody or by treatment of the L cells with actinomycin D. Kinase II activity was different from that of kinases I and III in that it showed Ca-dependence in the absence of Mg²⁺, was inhibited in activity by the SH binding agent N-ethylmaleimide, and could use the cellular enzymes RNase A and hexokinase as substrates. The described protein kinases could play an important role in mediation of IFNγ effects, particularly where phosphorylated enzyme substrates were shown to have altered activity.     

Interleukin 2 regulates immune interferon (IFN gamma) production by normal and suppressor cell cultures

A Lyt-2+ lymphocyte is responsible for immune interferon (IFN gamma) production in mitogen-stimulated mouse spleen cell cultures. A Lyt-1+ helper cell is required for the induction of IFN gamma. Interleukin 2 (IL 2) can specifically replace the Lyt 1 cell requirement. IL 2 will also abrogate suppressor cell inhibition of IFN gamma production. IFN gamma production appears to be regulated by a dynamic interaction between helper cells (source of IL 2), suppressor cells (absorption of IL 2?) and IFN gammna-producer cells.     

Antibody to staphylococcal enterotoxin A-induced human immune interferon (IFN gamma)

Antiserum to human gamma interferon (IFN gamma) was produced in rabbits immunized with partially purified (10(4.8) to 10(6.2) antiviral U/mg protein) staphylococcal enterotoxin A-induced IFN gamma. Staphylococcal enterotoxins, phytohemagglutinin M, concanavalin A, and pokeweed mitogen-induced antiviral activity in human leukocyte cultures was neutralized to undetectable levels by the antiserum. However, human leukocyte interferon (IFN alpha), human fibroblast interferon (IFN beta), and mouse interferons were not neutralized by the antiserum. After determining the antiserum was specific for IFN gamma and did not neutralize other known types of interferon, it was used with antibody to human IFN alpha to demonstrate the type(s) of interferon stimulated by some new inducers and antigens. Galactose oxidase- and calcium ionophore-induced interferons were neutralized to undetectable levels by the antiserum to IFN gamma. Interferon produced in leukocyte cultures from tuberculin-negative individuals stimulated with tuberculin-purified protein derivative or old tuberculin was IFN alpha, whereas interferon from tuberculin-positive individuals was a combination of alpha and gamma IFN. In addition, the antiserum neutralized the anticellular and natural killer cell enhancement activities of IFN gamma preparations. The specificity of this antiserum for IFN gamma indicates that it is an additional, powerful tool for identifying and classifying known and new interferons produced in vitro or in vivo and for investigating the role(s) of IFN gamma during the course of infectious, neoplastic, and autoimmune diseases.     

In vitro production of immune interferon by spleen cells of mice immunized with herpes simplex virus.

In Vitro production of Immune Interferon (IF) in response to Herpes Simplex Virus (HSV) antigen by sensitized spleen cells from C57B1/6 (B6) mice could be detected as early as 3 and for at least 20 days after ip infection of HSV. Maximal levels of IF were produced after 10 hr of culture, but there was no decay of activity when supernatants were sampled during the subsequent 3 days. The IF produced shared certain known properties of immune IF and was not neutralized by an antiserum against viral-induced (type I) IF. DBA/2 (D2) mice which are considerably more sensitive in vivo to HSV infection than B6 mice produced significantly lower amounts of immune IF in the in vitro test system regardless whether high or low doses of virus were injected. The same pattern of results was observed when resistant B6D2F1 hybrid mice were compared with $\text{A}\text{J}$ and Balb/c mice which are about as sensible to ip infection with HSV as DBA/2 mice in our laboratory. These results demonstrate a remarkable defect of in vitro cellular immunity in mice susceptible to a virus infection when compared with resistant mice. Conceivably, a similar defect may be of in vivo relevance.     

Cyclosporin A inhibits the production of gamma interferon (IFN gamma), but does not inhibit production of virus-induced IFN alpha/beta

The effect of cyclosporin A (CsA) on the production of gamma interferon (IFN gamma) versus IFN alpha/beta was studied using mouse and human lymphocytes and fibroblasts. Spleen cells from C57Bl/6 mice produced low but significant levels (40-60 U/ml) of IFN gamma after 2 to 3 days of culture with irradiated DBA spleen cells. The addition of CsA at concentrations as low as 0.1 microgram/ml completely inhibited (less than 10 U/ml) IFN gamma production in these cultures. High levels of IFN gamma (170-1200 U/ml) were produced when either C57Bl/6 spleen cells or Ficoll-Hypaque-purified human peripheral blood lymphocytes (PBL) were cultured with the T-cell mitogen staphylococcal enterotoxin A (SEA). The addition of CsA (0.1 microgram/ml) to these cultures also completely inhibited (less than 10 U/ml) IFN gamma production. This inhibition was shown not to be due to a change in the kinetics of IFN gamma production or to a change in the amount of SEA required for stimulation. IFN gamma production in SEA-stimulated mouse spleen cells was inhibited at 3 days of culture even when CsA was added at 24 or 48 hr postculture initiation. Thus, CsA inhibits IFN gamma production even when early events associated with lymphocyte activation have been allowed to take place. In contrast to IFN gamma production, IFN alpha/beta production by Newcastle disease virus (NDV)-infected mouse and human lymphocytes or fibroblasts was not inhibited by the addition of CsA (1 microgram/ml). CsA also did not block the action of IFN gamma or IFN alpha/beta since addition of CsA (1 microgram/ml) to reference IFN standards had no effect on their antiviral activity. Thus, CsA inhibits the production of IFN gamma by T cells but appears to have no effect on the production of IFN alpha/beta by virus-infected cells or on the antiviral action of already produced IFN gamma and IFN alpha/beta.     

Construction, purification, and characterization of new interferon gamma (IFN gamma) inhibitor proteins. Three IFN gamma receptor-immunoglobulin hybrid molecules.

Three efficient mouse interferon gamma (MoIFN gamma) inhibitors were constructed, which consist of the MoIFN gamma receptor (MoIFN gamma R) extracellular portion and constant domains of immunoglobulin (Ig) molecules. These are: 1) the constant domain of the mouse kappa chain, 2) the hinge region and the constant domains 2 and 3 of the mouse gamma 2a chain, and 3) the hinge region and the constant domains 2 and 3 of the human gamma 3 chain. The hybrid molecules were expressed in the mouse myeloma cell line J558L and recovered from the supernatants of cell cultures in one purification step. The proteins MoIFN gamma R-M gamma 2a and MoIFN gamma R-H gamma 3 form homodimers, whereas MoIFN gamma R-M kappa is a monomer. All three constructs inhibit the binding of radiolabeled MoIFN gamma to its receptor on L1210 cells. They are biologically active in vitro, neutralizing the action of MoIFN gamma in an antiviral activity assay. The fusions of Ig regions to the soluble MoIFN gamma R do not decrease the affinity of the binding site for the ligand. MoIFN gamma R-M kappa has about the same affinity as the soluble MoIFN gamma R and the cell surface receptor of L1210 cells in situ, which are also monomers, whereas the dimers MoIFN gamma R-M gamma 2a and MoIFN gamma R-H gamma 3 display a 5-10-fold higher affinity for MoIFN gamma than the monomeric molecules. This is best documented in the efficacy of the inhibitors to antagonize the antiviral activity of MoIFN gamma, as the dimeric constructs are about 10 times more active than MoIFN gamma R-M kappa and the soluble MoIFN gamma R. The hybrid constructs can be used as high efficiency MoIFN gamma inhibitors in mouse models of several pathological states in humans, where IFN gamma is thought to play a disease-promoting role.     

Mouse immune interferon enhances fibronectin production of elicited macrophages

By using mouse immune interferon (IFN-gamma) either produced by recombinant DNA technology or partially purified from a T cell lymphoma, L12-R4, we conclusively demonstrated that mouse IFN-gamma enhances fibronectin synthesis and secretion of proteose-peptone-elicited macrophages, but not of normal macrophages. The enhancing activity of both IFN-gamma preparations was acid-labile and was neutralized by a rabbit antiserum made against native IFN-gamma. In addition, IFN-gamma induced the disappearance, as revealed by immunofluorescence studies, of the characteristic fibronectin streaks from the cell surface of elicited macrophages. Taking into account the opsonizing activity of fibronectin, these findings offer some explanations for the IFN-gamma-mediated increase of macrophage phagocytosis and tumor cell killing.     

Reciprocal changes in interferon production and immune responses of mouse spleen cells fractionated over columns of insolubilized conjugates of histamine.

Cultures of spleen cells from unimmunized and immunized BALB/c mice can support both direct and indirect anti-sheep erythrocyte plaque-forming responses. The responsiveness of spleen cells to this thymus-dependent sheep cell antigen can be altered by fractionation of the cells over insolubilized conjugates of histamines (H-R-S). Cells that do not adhere to H-R-S (i.e., those that pass through the columns) produce a significantly greater plaque-forming cell response than do non-chromatographed cells or cells that have passed through a control column of rabbit serum albumin-Sepharose (R-S). In contrast, the direct plaque-forming cell response of the same culture to Salmonella typhimurium lipopolysaccharide (a T-lymphocyte-independent antigen) was not significantly different in any of the groups of cells tested. In addition, spleen cell filtration over H-R-S also resulted in a significant increase in the blastogenic response to phytohemagglutinin as well as a significantly lower production of interferon in response to phytohemagglutinin. The possibility that suppressor cells (which were shown to adhere to H-R-S) produce their inhibitory effect via interferon is discussed.     

Messenger RNA of mouse immune interferon (MuIFN-gamma)

A lysate of $\text{Staphylococcus aureus}$ ($\text{S. aureus}$) induced high-titered (10^3.6 ～ 10^4.6 units/ml) mouse immune interferon (MuIFN-γ) in spleen cell cultures. mRNA was extracted from such cells, purified by oligo(dT) cellulose chromatography and sucrose gradient centrifugation, and injected in $\text{Xenopus laevis}$ oocytes. Fractions containing RNA of 15 to 16 S gave rise to IFN activity, that was characterized as IFN-γ by virtue of its acid lability, neutralization by antiserum to partially purified MuIFN-γ, and lack of neutralization by antiserum to NDV-induced L-cell IFN.     

Effect of hydrocortisone and bacterial lipopolysaccharide on colony-stimulating activity production from mouse marrow adherent cells, spleen cells and peritoneal macrophages in vitro

The effect of hydrocortisone (HC) on colony-stimulating activity (CSA) production from mouse bone marrow adherent cells, spleen cells and peritoneal macrophages with or without bacterial lipopolysaccharide (LPS) stimulation was studied. CSA in the supernatant from bone marrow adherent cells incubated with HC was found to be five times higher than CSA from cultures without LPS stimulation. In contrast, the CSA production by spleen cells and peritoneal macrophages were significantly suppressed by HC in both LPS-stimulated and non-stimulated cultures. These studies suggest that the effect of HC on CSA production was quite different depending on the target cells.     

In vivo and in vitro macrophage activation induced by IFN gamma spontaneously released by spleen cells from tumor bearing mice.

Peritoneal macrophages obtained from Lewis Lung carcinoma (3LL) tumor bearing mice release high amounts of soluble factors such as C3,H2O2 and lysosomal enzymes but fail to exert cytotoxic activity on tumor cells. In the present work we show that they acquire this property and become fully activated after in vitro incubation with supernatants derived from cultures of splenocytes from tumor bearing syngeneic mice. The presence of IFN gamma in the above supernatants was detected by immunoblotting analysis and by bioassay. The role played by IFN gamma in macrophage activation was investigated.     

Influenza virus-induced interferon production in mouse spleen cell culture: T cells as the main producer.

Interferon production by spleen cells from unimmunized C3H mice challenged in vitro with influenza virus AO/PR8 was investigated. Glass-nonadherent cells (lymphocytes) produced significant levels of interferon, although cocultivation of glass-adherent macrophages was needed for optimal production. Treatment of the cells with antithymocyte serum and complement markedly reduced the interferon production. When glass-nonadherent cells were fractionated on a nylon wool column, the T-cell-enriched fraction consistently produced more interferon than the B-cell-enriched fraction. It is concluded that T cells are an important producer of interferon in spleen cell cultures from normal mice upon challenge with influenza virus, although non-T cells (macrophages and B cells) also may produce interferon under suitable conditions.