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.     

Relative Antiviral Resistance Induced in Homologous and Heterologous Cells by Cross-Reacting Interferons

Human cells incubated with human interferon become more resistant to vesicular stomatitis virus (VSV) than to Semliki Forest virus (SFV); monkey cells treated with monkey interferon become more resistant to SFV than to VSV. However, monkey cells incubated with human interferon developed relative antiviral activity identical to that induced by homologous interferon, and human cells developed characteristic human interferon-induced relative antiviral activity when exposed to monkey interferon. Therefore, cross-reacting interferons induce the relative antiviral activity characteristic of the interferon-treated cell rather than the cell of the interferon's origin. This relationship supports the hypothesis that interferon is not itself antiviral but rather induces cells to develop their own antiviral activity.     

Beta and gamma interferons act synergistically to produce an antiviral state in cells resistant to both interferons individually.

We showed previously that the mouse fibroblastoid cell line Ltk-aprt- is resistant to the antiviral effects of beta interferon. This lack of response reflects a partial sensitivity to the interferon that is accompanied by a failure to activate expression of several interferon-regulated genes, although certain other genes respond in a normal manner. We show here that Ltk-aprt- cells were also unable to establish an antiviral state and to activate expression of 2,5-oligo(A) synthetase when treated with gamma interferon. Strikingly, however, treatment with a combination of beta interferon and gamma interferon provided complete protection against viral replication. Although the cells were completely insensitive to up to 250 U of the interferons per ml added singly, essentially complete protection from viral cytopathic effects was achieved when as little as 10 U of each of the interferons per ml were combined. Expression of 2,5-oligo(A) synthetase was also sensitive to this synergistic effect. Activation of an antiviral state could also be achieved by sequential treatment, first with gamma interferon and then with beta interferon. Partial protection against viral replication could be achieved by pretreatment with gamma interferon for as little as 1 h before incubation with beta interferon and could be blocked by the addition of specific antibodies or by cycloheximide, indicating that gamma interferon induces the synthesis of a protein which can act synergistically with a signal produced by the beta-interferon receptor. We suggest that Ltk-aprt- cells suffer from defects in one or more components of the gene activation pathways for both type I and type II interferons. Nonetheless, gamma interferon is able to activate the expression of a gene encoding a protein required for signal transduction. This protein acts synergistically with a transient signal produced in response to beta interferon, thereby activating the expression of a further group of genes.     

Tumor Necrosis Factor Alpha Exerts Powerful Anti-Influenza Virus Effects in Lung Epithelial Cells

Previous studies have associated influenza virus-induced expression of inflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), with influenza pathogenesis in the human respiratory tract and have suggested that alpha and beta interferons are the first cytokines recruited to counteract such infection. However, we report here that TNF-alpha has powerful anti-influenza virus activity. When infected with influenza virus, cultured porcine lung epithelial cells expressed TNF-alpha in a dose-dependent manner. Expression of TNF-alpha was induced only by replicating virus. TNF-alpha showed strong antiviral activity against avian, swine, and human influenza viruses, and the antiviral effect of TNF-alpha was greater than that of gamma or alpha interferon. These findings suggest that TNF-alpha serves as the first line of defense against influenza virus infection in the natural host.     

Additive and synergistic effects of a novel antiestrogen, toremifene (Fc-1157a), and human interferons on estrogen responsive MCF-7 cells in vitro

The effect of human interferons alpha and gamma alone and in combination with a novel antiestrogen toremifene were studied in vitro using MCF-7 cell line, an estrogen receptor positive and antiestrogen sensitive cell line. The effects were evaluated by a simple bioluminescence method with which the number of living cells was obtained as cellular adenosine triphosphate (ATP) content. The growth of MCF-7 cells was inhibited both by interferon alpha and interferon gamma. At least additive effect was evident when the cells were exposed to combination of interferons and toremifene: the combination was additive with interferon gamma + toremifene and synergistic with interferon alpha + toremifene. The combination of toremifene and interferons may have clinical importance.     

Induction of cell surface antigens on a lymphomatous cell line by gamma interferon

The interferons are known to induce the expression of cell surface determinants. Gamma interferon, in particular, has been shown to enhance class II antigens (DR) on the cell surface. We used this gamma interferon to induce beta 2-microglobulin (beta 2 mu) and a minor, sex related transplantation antigen called H-Y on the surface of B lymphoma cells. The antitumor effect of interferon could thus be at least twofold: an antiviral effect already known and an increase of intercellular recognition (by cell surface marker enhancement); allowing the tumor cells to be "seen" better by cells of the immune system.     

Antiviral and antiproliferative activity of human interferons and their induction of 2',5'-oligoadenylate synthetase

Native preparations of alpha, beta and gamma-interferons as well as recombinant beta-interferon and purified leukocyte alpha-interferon and purified leukocyte alpha-interferon exert antiviral and antiproliferative activity in CaOv cells. Native interferon preparations were shown to be more antiproliferative than purified interferons per unit of antiviral activity (with EMC as well as with less susceptible VSV used as test viruses). It was shown that level of 2'5' oligoadenylatesynthetase activity induction in general correlates with antiproliferative and pronounced antiviral activity of interferons, besides that, the earlier (by 11 hours) induction of the enzyme activity by beta-interferon correlates with more rapid expression of antiproliferative effects by this interferon in comparison with that of alpha-interferon, the latter inducing the peak of enzyme activity by 24 hours.     

Interferon gamma does not induce antiviral resistance in T lymphocytes

We compared the activity of human recombinant alpha and gamma interferons (IFNs) on normal T lymphocytes and various T cell lines. IFN gamma, unlike IFN alpha, did not promote the antiviral state in these cells, or induce the activity of 2'-5' oligoadenylate synthetase. The lack of antiviral effect was observed using an RNA virus (VSV) and a DNA virus (HSV, type 1) as challenger viruses.     

Differential effects of pure human alpha and gamma interferons on fibroblast cell growth and the cell cycle

Pure human alpha and recombinant gamma interferons had differential effects on two strains of fetal lung fibroblasts in vitro. Alpha interferon had little effect on long-term cell growth, whereas gamma interferons, both glycosylated and non-glycosylated, were cytotoxic. However, when synchronized cells were studied, alpha interferon prolonged both G1 and S + G2 phases of the cell cycle, whereas gamma interferon only affected the G1 phase.     

Differential antiproliferative activities of IFNs alpha, beta and gamma: kinetics of establishment of their antiproliferative effects and the rapid development of resistance to IFNs alpha and beta

Interferons have been recognized to have potent in vitro antiproliferative activities in mouse and human systems. To further investigate the kinetics of development of interferons' antiproliferative activities, mouse B-16 melanoma cells were treated with MuIFN-alpha, MuIFN-beta or MuIFN-gamma for various initial periods of time during an 8 day cloning assay. With MuIFN-alpha and MuIFN-beta treatments, maximal expression of antiproliferative activity was attained with 2 to 4 days of interferon treatment. In contrast, with MuIFN-gamma treatment, expression of antiproliferative activity increased with progressively longer periods of time of MuIFN-gamma treatment. These results suggested that B-16 melanoma cells were initially sensitive to all three of the interferons but rapidly became resistant to MuIFN-alpha and MuIFN-beta after 2 to 4 days of treatment. This suggestion was confirmed by cell growth kinetics experiments. The cells which were resistant to the antiproliferative activity of the MuIFN-alpha remained sensitive to the antiviral activity of MuIFN-alpha, suggesting that MuIFN-alpha and MuIFN-beta regulate their antiviral and antiproliferative responses via different mechanisms. The cells which were resistant to the antiproliferative activities of MuIFN-alpha and MuIFN-beta remained sensitive to MuIFN-gamma, suggesting that they were not generally resistant to antiproliferative effects. The cells which were resistant to the antiproliferative activities of the interferons gradually lost their resistance with a half-life of 11 days when they were cultured in the absence of interferons. The differential antiproliferative actions of alpha, beta and gamma interferons observed with murine B-16 melanoma were confirmed in the human system with G-361 melanoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of interferons produced by peripheral blood mononuclear cells from healthy subjects in response to Corynebacterium parvum or poly I: poly C

The biological significance of acid labile interferon alpha is presently unknown. We examined the putative production of acid labile interferon in vitro from human peripheral blood mononuclear cells induced with Corynebacterium parvum or poly I: poly C. Both agents induced up to 1200 IU/ml interferon, and the interferon was 80 to 90% acid labile. The interferons were typed by antibody neutralization of their antiviral activity. Contrary to previous reports, C. parvum induced predominantly interferon gamma, which is normally acid labile, whereas poly I: poly C induced an acid labile interferon alpha activity with characteristics similar to those of acid labile interferon alpha reported in serum in certain human diseases.     

Effect of in vitro interferon treatment on the rapid clearance of murine leukemia cells in vivo.

Previous work showed that interferon (IFN) can protect target cells from NK mediated lysis in vitro. In the present study we investigate the effect of IFN alpha/beta or IFN gamma treatment of three different murine leukemia cell lines. For this purpose FLC-745 (susceptible to the antiproliferative activity of IFN alpha/beta and gamma), FLC-3C18 (IFN alpha/beta -resistant and IFN gamma - susceptible) of DBA/2 origin and EL-4 (IFN alpha/beta - susceptible and IFN gamma - resistant) leukemia of C57B1/6 origin were treated with IFN alpha/beta or gamma in vitro and assayed for their susceptibility to natural resistance measured in vivo as organ rapid clearance 4 hr after iv injection into syngeneic mice. Using young or Poly I:C stimulated hosts, but not mice with low levels of natural resistance (i.e. older animals or mice treated with cyclophosphamide), slower elimination of treated cells was observed with: (a) FLC-745 cells treated with IFN alpha/beta and IFN gamma and (b) FLC 3C18 treated with IFN gamma. Such a delayed clearance was not observed with: (a) FLC-3C18 cells treated with IFN alpha/beta and (b) EL-4 leukemia cells preincubated with IFN alpha/beta or IFN gamma. These results suggest that under selected conditions IFNs can protect leukemic cells from in vivo natural reactivity.     

Natural killer (NK) cells as a responder to interleukin 2 (IL 2). II. IL 2-induced interferon gamma production

In the accompanying paper, we showed that natural killer (NK) cells were a major population in the naive spleens of normal mice that responded directly to a T cell growth factor, interleukin 2 (IL 2), and clonally replicated without other stimulating agents. The cloned cells growing in IL 2 showed a potent NK activity against several NK targets without addition of an NK-activating agent, interferon (IFN). In the present study, therefore, we examined whether these cloned NK cells on their own produced IFN. It was found that all NK clones growing in IL 2 produced IFN in the culture fluids. The titers of IFN produced in the IL 2-containing media correlated well with the number of growing cells. With the culture in the absence of IL 2, neither cell growth nor IFN production could be detected. Addition of Con A into the culture in the IL 2-free media showed no IFN production. The antiserum neutralizing IFN alpha and IFN beta failed to significantly neutralize IFN produced by NK clones. Treatment with either a pH of 2.0 or antiserum neutralizing mouse IFN gamma resulted in a marked reduction of IL 2-induced NK IFN, indicating that a major part of IFN produced was IFN gamma. These results indicate that IL 2 stimulates NK clones to proliferate, accompanied by IFN gamma production. The results also show that an NK clone, when stimulated with Sendai virus, produced a type 1 IFN (IFN alpha and/or IFN beta), suggesting that murine NK cells can produce both type 1 (alpha and/or beta) and type 2 (gamma) IFN, depending on inducers.     

Susceptibility of different leukemic cell lines to the anticellular and antiviral effects of interferons

A total of 18 different types of human leukemic cell lines were tested for their susceptibility to the anticellular and antiviral effects of interferons (IFNs) alpha, beta and gamma. In general, only the three myelogenous leukemic cell lines U937, KG-1 and HL-60 were found to be highly susceptible to the anticellular effect of the different IFNs while cells of the other lineages were relatively resistant. In order to determine whether the cell lines were sensitive to the antiviral effects of IFNs, the cells were first screened for their ability to support the replication of vesicular stomatitis virus (VSV), sindbis virus (SBV) and semliki forest virus (SFV). Unexpectedly, only three cell lines--Raji, K562 and U937 were highly susceptible to SFV while other cell lines were relatively refractory to all three viruses. Using SFV as indicator virus, the antiviral activity of all IFNs could be detected in all three cell lines and their relative efficiency was in the order of alpha greater than beta greater than gamma. The significance of these results were discussed.     

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.     

Effects of butyrate on induction and action of interferon

We have identified two different and independent effects of sodium butyrate on induction and action of interferon. In the monkey cell line, GL-V3, simultaneous treatment with interferon and butyrate strongly reduced the antiviral activity of the interferon preparation, Whereas addition of butyrate before interferon or after establishment of the antiviral state had no effect. Interferon production induced by Sendai virus was also reduced by simultaneous treatment with butyrate, but pretreatment resulted in marked enhancement of interferon yields. Whereas the inhibitory effects of simultaneous butyrate treatment were also observed in human (WISH) and bovine (MDBK) cells, pretreatment with butyrate in these cells had no effect on interferon yields.     

Effects of alpha, beta and gamma recombinant interferons on CEA production from HT-29 human colon adenocarcinoma cell line

The human colon adenocarcinoma derived cell line HT-29 is a good in vitro model for the study of CEA production and release under various experimental conditions. Many studies indicate that CEA secretion is correlated with cell proliferation and seems to depend on the growth conditions and differentiation characteristics induced by the culture medium. The present study demonstrates that recombinant interferons alpha, beta and gamma (rIFN alpha, rIFN beta, rIFN gamma) can modify CEA production and release by HT-29 cell-line. rIFN gamma in particular causes an enhancement of CEA production and release in the culture medium. This dose-depending effect is in some way correlated to cell growth inhibition since the enhancement of CEA expression in the interferon treated cells is evident in the presence of a reduction in cell proliferation. The activity of rIFN alpha and rIFN beta on CEA release is much less remarkable than that demonstrated by rIFN gamma, and is probably only due to the fact that HT-29 colon adenocarcinoma cells respond poorly to the effects of rIFN alpha and rIFN beta at the doses we used. These findings suggest that CEA production, expression and release can be modulated in a variety of ways under the influence of different rIFN treatment and this situation must be taken into account in immunodiagnostic and immunotherapeutic applications of anti-CEA monoclonal antibodies in the cancer patient.     

Importance of interferons in recovery from mousepox.

Gamma interferon is shown to be critical in recovery of C57BL/6 mice from mousepox. Anti-gamma interferon treatment of mice infected in the footpad with ectromelia virus resulted in enhanced spread to and efficient virus replication in the spleen, lungs, ovaries, and, especially, liver. All treated, infected mice died within a mean of 7 days, 2.5 days earlier than mice with severe combined immunodeficiency that were given a comparable infection. On the other hand, alpha interferon appeared not to have a major role in controlling virus replication in tissues examined, and beta interferon was important for virus clearance in the liver and ovaries but not the spleen. Either anti-alpha, beta interferon or anti-beta interferon antibody therapy resulted in only 25% mortality. Infected control mice survived but showed persistence of ectromelia virus at the site of infection (the footpad) and transient presence of the virus in the spleen, liver, lungs, and ovaries and in the fibroreticular but not lymphoid cells of the draining popliteal lymph node. Depletion of gamma interferon but not alpha and/or beta interferon resulted in a significant reduction in the numbers of splenic T (especially gamma delta-TCR+), B, and Mac-1+ cells, although the proportion of Mac-1+ cells in the spleen increased compared with control values. Depletion of alpha, beta, or gamma interferons did not severely affect the generation of virus-specific cytotoxic T-lymphocyte responses or natural killer cell cytolytic activity. This study, in which a natural virus disease model was used, underscores the crucial importance of gamma interferon in virus clearance at all stages of infection and in all tissues tested except the primary site of infection, where virus clearance appears to be delayed.     

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.     

Additive and synergistic antitumor effects with toremifene and interferons

MFC-7 cells were exposed to toremifene, human alpha and gamma interferons and combinations of them in vitro. Growth of the cells was followed by ATP bioluminescence method. Rats bearing DMBA-induced tumors were treated with toremifene, rat gamma interferon and their combination daily for five weeks. The growth of the tumors was followed by palpation weekly. Toremifene and interferons inhibited the growth of MCF-7 cells. Interferons alpha and gamma were additive; toremifene and interferons were additive or at the best synergistic. Toremifene inhibited the growth of DMBA-induced tumors. Rat gamma interferon alone had no clear effect on the tumor growth. Combination of toremifene and gamma interferone was the most effective treatment and did not show any detectable toxicity. Toremifene and interferons have interesting interactions. Clinical studies using the combination might be warranted.