In vivo and in vitro induction of (2'-5') oligoadenylate synthetase by human interferons in leukocytes from healthy donors and patients with renal cancer and hairy cell leukemia

The effect of in vivo administered interferon-alpha (IFN-alpha) on 2-5-oligoadenylate (A) synthetase activity of peripheral blood mononuclear cells (PBMC) was compared in patients with hairy cell leukemia and renal cell cancer. Basic levels of this enzyme varied from donor to donor, but mean levels were not significantly different in patients with renal cell cancer or hairy cell leukemia compared to healthy donors. After a single injection of 3 x 10(6) IU IFN, these basic levels rose 2- to 8-fold within 12-24 h post-injection and reverted to pretreatment levels after 48 h. The extent of this in vivo stimulation by IFN-alpha was similar in patients with hairy cell leukemia and renal cell carcinoma, and was correlated with down-regulation of IFN-alpha receptors. The in vitro effects of IFN-alpha, -beta and -gamma were compared after 18 h treatment with 10, 10(2) and 10(3) IU/ml of each IFN. Unlike IFN-alpha and -beta, IFN-gamma did not induce 2-5 A synthetase activity in either normal PBMC or hairy cells; these results were related to the effects of the three IFN on proliferative response of normal PBMC to phytohemagglutinin. Our data support the idea that 2-5 A synthetase activity is a marker of biological response to interferon treatment in human cancers.     

Sensitivity of human germ-cell-tumor cell lines to human interferons

We examined the sensitivity of four human germ-cell-tumor cell lines exhibiting different stages of differentiation to human interferons (IFNs) in vitro. The cell lines were derived from two embryonal carcinomas (NEC 8 and NEC 14), a choriocarcinoma (IMa), and a yolk-sac tumor (HUOT). Treatment with poly I:C induced IFN production in IMa and HUOT cells, but not in NEC-8 and NEC-14 cells. In the two embryonal-carcinoma cell lines, the addition of IFN-alpha, IFN-beta, and IFN-gamma did not prevent infection by vesicular stomatitis virus and encephalomyocarditis virus. Also, in these two lines, 2-5A synthetase was not induced by the addition of IFN-alpha. In contrast, both IMa and HUOT showed sensitivity to the antiviral action of IFN-alpha and IFN-beta against the two viruses, and 2-5A synthetase was induced by IFN-alpha. IFNs added at doses of up to 1000 IU/ml had no antiproliferative effect on NEC 8, NEC 14, and HUOT, whereas colony formation by IMa cells was greatly suppressed by all three forms of IFN. These results indicate that the production of and sensitivity to IFN are developmentally regulated and are related to the level of differentiation of human germ-cell stem cells.     

Activation of 2'',5''-oligoadenylate synthetase activity on induction of HL-60 leukemia cell differentiation.

A 27-fold increase in 2',5'-oligoadenylate synthetase activity, an enzyme associated with the antiproliferative actions of interferon (IFN), was observed after treatment of HL-60 human leukemia cells with dimethyl sulfoxide (DMSO), an inducer of granulocytic differentiation of the cells. Enzyme activity was elevated after 24 h of exposure to DMSO, was maximal at 48 hours, and declined thereafter. A comparable increase was observed after treatment with 1 U of alpha interferon (IFN-alpha) per ml or 8 U of beta interferon (IFN-beta) per ml. Elevated levels of expression of other IFN-inducible genes, including type I histocompatibility antigen (HLA-B) mRNA and 2',5'-oligoadenylate phosphodiesterase activity, were also observed with DMSO treatment. DMSO-treated HL-60 cells had an increased amount of a 1.8-kilobase mRNA for oligoadenylate [oligo(A)] synthetase when compared with that of control cells; both DMSO- and IFN-treated HL-60 cells also expressed 1.6-, 3.4-, and 4.3-kilobase mRNA. The increase in both oligo(A) synthetase activity and mRNA levels was inhibited by polyclonal antiserum to human IFN-alpha; however, no IFN-alpha mRNA could be detected in the cells. Antiserum to IFN-beta or gamma interferon (IFN-gamma) had no effect on oligo(A) synthetase expression or activity nor was there any detectable IFN-beta 1 or IFN-beta 2 mRNA in the cells. The anti-IFN-alpha serum did not block the elevation of HLA-B mRNA in DMSO-treated cells. These observations suggest that the increased expression of oligo(A) synthetase in DMSO-treated cells may be mediated by the release of an IFN-alpha-like factor; however, the levels of any IFN-alpha mRNA produced in the cells were extremely low.     

Expression of interferon-induced genes in different tissues of mice.

In vivo responses to interferon (IFN) in mice were determined by measuring the steady-state levels of induced mRNAs following injection of IFN and poly(I)-poly(C). With cDNA probes for mouse 2'-5' oligoadenylate synthetase (2-5A synthetase) and 1-8, constitutive expression of the corresponding mRNA was detectable in different organs of normal C3H/He mice. These mRNA levels were increased by as much as 15-fold over control levels in various tissues, including the brain, after IFN and poly(I)-poly(C) treatment, coincident with increases in 2-5A synthetase enzyme activity. The basal activity level of this enzyme could be reduced in normal mice by treatment with anti-mouse IFN (alpha + beta) antibody. This treatment also reduced the levels of 2-5A synthetase and 1-8 mRNAs. Thus, physiological levels of circulating IFN maintain elevated levels of IFN-induced mRNAs in mice. Furthermore, changes in 2-5A synthetase enzyme activity reflect the changes in gene expression in vivo.     

Isolation and characterization of an interferon-resistant cell line deficient in the induction of (2''-5'')oligoadenylate synthetase activity.

To screen for cells with different sensitivities to interferon (IFN), NIH 3T3 mouse fibroblasts were subcloned and examined for their response to IFN treatment. Of 30 clones tested, 2 appeared to be relatively resistant to IFN, since the replication of both vesicular stomatitis virus and mengovirus was not inhibited, even in the presence of 1,000 U of IFN per ml. One resistant (A10) and one sensitive (A5) clone were further analyzed. In both clones, murine leukemia virus replication was equally inhibited by IFN, indicating the presence of functional receptors for IFN in the resistant clone. Using the (2'-5')oligoadenylate (2-5A) radiobinding assay, we could demonstrate that both clones contained the RNase L protein. Furthermore, this enzyme appears to be active, since a similar reduction in the rate of protein synthesis was evident after the introduction of exogenous 2-5A to the cells. We also analyzed the activity of another enzyme in the 2-5A pathway, namely, 2-5A synthetase. In the sensitive cells (A5), the induction of enzyme activity was proportional to the IFN concentration used, reaching a maximum of more than a 10-fold increase over the background of untreated cells. However, little if any induction over the basal activity was observed in the resistant cells (A10) when similar doses of IFN were used. It is thus probable that the lack of induction of 2-5A synthetase activity by IFN in A10 cells is at least partly responsible for their relative resistance to IFN treatment.     

Persistent augmentation of natural-killer-and T-cell-mediated cytotoxicity in peripheral blood mononuclear cells pulsed in vitro with high-dose recombinant interleukin-2 prior to culturing with a low maintenance dose

The toxicity of high-dose recombinant interleukin-2 (rIL-2) treatment limits its use in tumour therapies. This paper describes in vitro studies of whether a single, peak rIL-2 dose, followed by low maintenance doses, could enhance the cytotoxic potential of peripheral blood mononuclear cells (PBMC) without inducing a significant sustained release of secondary cytokines, known to contribute to undesirable side-effects of therapy. Pre-pulsing of PBMC with high-dose rIL-2 (16000 IU/ml for 30 min), followed by low-dose (5 IU/ml) maintenance culturing, was found to induce persistent augmentation of cytotoxic activity towards natural-killer(NK)-sensitive and-insensitive tumour targets, as well as increased T-cell-mediated target cell killing. Under these conditions the level of killing was as high as that achieved by higher maintenance doses (20–100 IU/ml). Although not reflected by overexpression of cell surface markers, enhanced activation of cytotoxic capacities by high-dose pre-pulsing remained clearly apparent for at least 12 days of culture. Increased secondary cytokine production (tumour necrosis factor, interleukin-6 and interferon ) was only evident during the first 24–72 h after pulsing, and not at later stages of culturing at the low maintenance dose of 5 IU rIL-2/ml. These results may warrant a human phase-1 B study to investigate the in vivo effect of high-dose prepulsing, followed by low-dose maintenance.     

VIP induces in HT-29 cells 2'5'oligoadenylate synthetase and antiviral state via interferon beta/alpha synthesis.

Vasoactive intestinal peptide (VIP), composed of 28 amino acids, is a multifunctional neurotransmitter. We have demonstrated here that its action on human transformed colonic epithelial (HT-29) cells is mediated through the induction of interferon (IFN) synthesis. We have found that these cells have a functional receptor for IFN alpha 2; binding was specific to either IFN alpha 2 or IFN beta but not to IFN gamma. VIP induced the 2'5'oligoadenylate synthetase (2'5'A synthetase) and the antiviral state with the same efficiency as poly (I).poly (C). The induction of 2'5'A synthetase activity required cellular RNA and protein synthesis, and the maximum induction occurred with 10(-7) M VIP at 24 h. VIP, like some IFN inducers, induced the synthesis of the 70 hsp which, however, preceded the expression of 2'5'A synthetase. VIP treatment caused the induction and secretion of IFN, having a titer value of 32 international units/ml. This IFN has been identified as type beta/alpha, because both 2'5'A synthetase and the antiviral activities were abolished by anti-human IFN beta/alpha antibodies, but not by anti-IFN gamma antibodies. Thus the pathway of VIP action on HT-29 cells may be outlined as 1) binding of VIP, 2) synthesis of 70 hsp, 3) induction of IFN synthesis and its secretion, 4) binding of the secreted IFN to cell surface receptors and 5) turning on the induction of 2'5'A synthetase and antiviral activities.     

Tumor necrosis factor-alpha induces vitamin D-1-hydroxylase activity in normal human alveolar macrophages.

The induction of 1-hydroxylase in alveolar macrophages by tumor necrosis factor-alpha (TNF) was examined in view of recent evidence suggesting that local production of 1,25-(OH)2D3 may play a role in the regulation of immune functions. Incubation of pulmonary alveolar macrophages from normal human subjects with recombinant TNF caused a 2- to 10-fold increase in 25-hydroxyvitamin D3-1-hydroxylase activity. The dose-response curve was linear over the range 0.05-5.0 IU/ml, and no further increase was seen at higher concentrations. The increase in 1-hydroxylase activity was present after 12 h and reached a maximum after 3 days. The effect of TNF was inhibited in a dose-dependent manner by the presence of 1,25(OH)2D3 (10(-10)-10(-8) M) in the incubation media for 5 days but was unaffected by 10(-9) M 1,25(OH)2D3 after 12 h. The enhancement of macrophage 1-hydroxylase activity by TNF was comparable to that induced by gamma interferon (IFN) but the effects of maximal doses of both agents were not additive. The presence of antibody to TNF resulted in a 76% inhibition of TNF-induced 1-hydroxylase but had no significant effect on IFN-induced 1-hydroxylase activity.     

Influence of glucagon, 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate and triamcinolone on the arginine synthetase system in perinatal rat liver

1. The administration of triamcinolone (19-190mug/animal) to postnatal rats increased the arginine synthetase system activity 1.2-2.5-fold above control values 24h after exposure to the hormone. Cortisol (hydrocortisone), however, increased the arginine synthetase system activity only when larger (190mug/animal) or repeated daily doses were given. Glucagon (100mug/animal) stimulated arginine synthetase system activity only after the second postnatal day. None of these agents increased the activity in 19.5-21.5-day foetuses after intrauterine administration. 2. The viability of foetal rat liver explants maintained in organ culture for up to 54h was validated both by ultramicroscopic examination and by incorporation of radioactive leucine and orotic acid. 3. In organ cultures of foetal rat liver explants (18.5 days to term), triamcinolone (20mug/ml of medium) evoked a 2.8-4.3-fold increase after 24h of incubation. This increase was completely inhibited by actinomycin D (25mug/ml) or cycloheximide (10mug/ml). Cortisol (5-50mug/ml) or glucagon (0.067-67mug/ml) also increased the arginine synthetase system activity above the respective control values, but there was no increase in activity with insulin (0.05-0.25i.u./ml). 4. Maximum concentrations of glucagon (67mug/ml), dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate) (0.1mm) and triamcinolone (20mug/ml) incubated for 24h with foetal rat liver explants each produced between a two-and three-fold increase in the activity of the arginine synthetase system. Combinations of maximum amounts of glucagon and the cyclic nucleotide did not produce a greater effect than either agent alone. However, the combination of dibutyryl cyclic AMP with triamcinolone appeared to produce somewhat less than additive effects. 5. The effects of the cyclic nucleotide and triamcinolone were evident after 12h of incubation and increased steadily throughout the 24h of observation. This time-course of increased enzyme activity is very much slower than that reported for the induction of other enzymes in explant cultures of foetal rat liver.     

Enhancement of interferon-induced 2–5 oligoadenylate synthetase activity by retinoic acid in human histiocytic lymphoma U937 cells and WISH cells

The effect of retinoic acid (RA) on the level of interferon (IFN)-induced 2-5 oligoadenylate (2-5A) synthetase activity was examined in human histiocytic lymphoma U937 cells and WISH cells** in order to ascertain the role of this polymerase in interaction between IFNs and RA. Cultures containing both IFNs (1-100 U/ml) and RA (0.1-10 microM) consistently had higher levels of enzyme activity than corresponding cells treated with IFN alone and this was true for all three types of IFNs in both cell lines. The potentiating effect of RA was dose- and time-dependent and under optimal conditions, the induction of the synthetase was synergistic between IFN-beta (10-100 U/ml) and RA (0.1-10 microM). Furthermore, pretreatment (but not posttreatment) with RA followed by subsequent treatment with IFNs preferentially induced higher levels of enzyme activity in U937 cells but not in WISH cells. In addition, our results indicated that the modulating effect of RA on IFNs did not involve interaction at the receptor level and the level of enhancement of 2-5A synthetase activity was not in parallel with either cell-growth arrest or promotion of differentiation. Lastly, the present study raises the possibility that interactions between IFNs and RA, in either a synergistic or antagonistic manner, may be mediated through amplification of the 2-5A system.     

Development of an Immunofluorometric, High-Capacity, Cell-Based Assay for the Measurement of Human Type I and Type II Interferons

We have developed a cell-based 96-well microtiter plate, high throughput assay for measuring both type I and type II interferon (IFN) activity on human cells. This assay makes use of a previously described IFN-specific reporter stably expressed in human HT 1080 cells. The induction of the reporter by IFN is determined by measuring the IFN-dependent expression of CD2 on the cell surface. The cytokine-induced expression of CD2 occurs within 48 h and is measured using a time-resolved fluorometric immunoassay. The limit of detection for type I IFN is.0.4 IU/ml. Interassay and intraassay coefficients of variation were 1.1% and 1.3% for the medium control (31 IU IFNb1b/ml), respectively. The limit of detection for type II IFN is.8 IU/ml, and the assay coefficients of variation are similar to those determined for type I IFNs. The level of sensitivity for this assay is comparable to other assays commonly used to measure IFN activity on cells. The current assay has an advantage over antiviral and antiproliferative assays, in that there is no requirement for the use of pathogenic virus or for determining viable cell numbers. The current assay is ideally suited for increasing sample screening and high-capacity automation, making it an excellent tool for drug discovery.     

Induction of (2'-5')oligoadenylate synthetase in serum-starved HeLa S3 cells by growth factors and its role in growth regulation

When serum-starved HeLa S3 cells were stimulated to proliferate by addition of fetal calf serum (FCS), (2'-5')oligoadenylate synthetase (2-5A synthetase) activity was induced. Although no interferon (IFN) activity was detectable in the HeLa S3 cell-conditioned culture medium after growth stimulation, addition of anti-IFN-beta monoclonal antibody inhibited both the expression of the 2-5A synthetase gene and the production of the enzyme, suggesting that endogenous IFN-beta was involved in 2-5A synthetase induction. Purified preparations of three growth factors, epidermal growth factor, platelet-derived growth factor, and insulin, also induced 2-5A synthetase through IFN-beta. When serum-starved HeLa S3 cells were treated with FCS, DNA synthesis was initiated synchronously, with peaks after 12 and 32 h, although the level of 2-5A synthetase reached a maximum after the first peak of DNA synthesis. Inhibition of 2-5A synthetase induction by anti-IFN-beta antibody enhanced the second, but not the first cycle of DNA synthesis. These results suggested that in HeLa S3 cells, after stimulation with growth factors the IFN/2-5A synthetase system played a role in cell growth negative regulatory mechanisms.     

2',5'-Oligoadenylate synthetase expression is induced in response to heat shock

Hyperthermia (45 degrees C) induced the synthesis of a characteristic heat-shock protein of 70,000 daltons (70 hsp) in Madin-Darby bovine kidney (MDBK) cells. In addition, subsequent to heat shock, there was a substantial increase in the 2',5'-oligoadenylate synthetase (2',5'-A synthetase) activity in both MDBK and human WISH cells. However, in contrast to 70 hsp synthesis, which reached its maximum 3 h after cell transfer from 45 to 37 degrees C, increase in 2',5'-A synthetase expression, conspicuous after 6 h, attained its maximum only 18 h after transfer. Another interesting observation is that, during recovery at 37 degrees C, the cells released into the medium heat-shock-induced factor(s) (HSIF) capable of inducing an increase in 2',5'-A synthetase activity in fresh MDBK cells. HSIF behaves as a polypeptide with a molecular weight of more than 5,000; it is relatively heat stable and sensitive to acidic treatment. HISF seems different from interferon (IFN) since: 1) no detectable antiviral state developed after infection in cells treated with HSIF; 2) antibovine IFN antibodies did not abolish the inducing capacity of HSIF; 3) IFN had an additive effect on the inducing capacity of HSIF, and 4) HSIF released from bovine cells induced a net enhancement of 2',5'-A synthetase activity in human WISH cells. The first three of these observations applied also to heat-shocked MDBK cells.     

In vitro antitumor effect of LAK cells and alpha interferon in combination on tumor cell lines.

In vitro antitumor effects of LAK cells and alpha-2b-Interferon (IFN) either alone or in combination were evaluated on NK resistant (K562) and NK sensitive (Namalwa, Raji) cell lines. Tumor cells were incubated with LAK cells for 4, 8 and 24 hours at a LAK: tumor cell ratio of 1:1, 10:1, 100:1, or with IFN for 48 and 96 h at the concentrations of 100, 1000, 10,000, 100,000 IU/ml. A clonogenic assay was utilised to enumerate residual cells after in vitro treatment. A positive correlation was found between tumor cell killing and effector: target ratio, IFN of 100:1 incubated for 4 h, and 100 IU/ml of IFN incubated for 48 h were further chosen. A synergistic effect was found when IFN was incubated before LAK cells or contemporarily, but not when IFN was incubated after LAK cells. These findings demonstrate that an additive or a synergistic effect in vitro can be obtained by adding the two agents in different sequences and suggest that a potential utility of LAK cells and IFN in vivo should be tested in clinical trials.     

Effect of interferon on the production of HBsAg and induction of an antiviral state in human hepatoma cell line PLC/PRF/5

The effects of human alpha and beta interferons (IFN) on the production of HBsAG by PLC/PRF/5 cells, an HBsAg-producing human hepatoma cell line, were studied in the exponential and stationary phases of cell growth. When exponential phase cells were treated with 100 or 1,000 U of IFN per ml for 48 hr. the amount of HBsAg in the culture medium decreased. The number of cells and the synthesis of DNA and proteins were also reduced by the IFN treatment. These results suggested that IFN did not affect the production of HBsAg specifically in exponential phase cells. When cells in the stationary phase were similarly treated with IFN, HbsAg production was not inhibited nor did the number of cells decrease. To examine the antiviral state induced by IFN in PLC/PRF/5, induction of 2'5'-oligo (A) synthetase and susceptibility to two kinds of viruses were examined. The 2'5'-oligo (A) synthetase activity was increased in an IFN-dose dependent manner. Susceptibility to vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) was decreased by treatment with 10 and 100 U of IFN per ml for 20 hr. It was concluded that IFN-alpha and IFN-beta induce 2'5'-oligo (A) synthetase and the antiviral state, but do not inhibit HBsAg production by PLC/PRF/5 cells.     

Effect of human interferons on cAMP phosphodiesterase activity in a culture of human ovarian carcinoma cells (CaOv)

Crude and purified human interferons of alpha type exerted 2 step inhibition of cAMP phosphodiesterase activity in CaOv cells: in 4 and 24 hours after cells treatment with interferon. The maximal inhibition was obtained in response to interferon doses 1200-2000 IU/ml. In contrast to natural interferons the human alpha 2 recombinant interferon (20-25000 IU/ml) did not inhibit the cAMP phosphodiesterase activity in CaOv cells.