Comparison of pharmacokinetic behaviors of two human interferons (Lb-IFN-alpha and Re-IFN-alpha A) in cynomolgus monkeys by 2'-5' oligoadenylate synthetase assay

Two human interferons (IFNs), natural lymphoblastoid IFN-alpha (Lb-IFN-alpha) and recombinant IFN-alpha (Re-IFN-alpha A) were compared for their induction of 2'-5' oligoadenylate (2-5A) synthetase and pharmacokinetic behaviors in cynomolgus monkeys. In in vitro experiments, the enzyme activity induced in an epithelial cell strain from human amniotic membrane (FL) cells was much higher than that in primary culture of cynomolgus monkey kidney (PMK) cells, and Lb-IFN-alpha induced higher levels of the enzyme than did Re-IFN-alpha A in both cells. In in vivo experiments, no difference was found in clearance from the blood between the two IFNs, but the half life of the enzyme induced in peripheral leukocytes by Lb-IFN-alpha was about twice longer than that by Re-IFN-alpha A, and the enzyme levels detected in various tissues were IFN-dose dependent. These results indicate that natural Lb-IFN-alpha is more effective in inducing 2-5A synthetase than Re-IFN-alpha A, and that the enzyme activity of various tissues as well as peripheral blood lymphocytes (PBL) increased in the cynomolgus monkeys administered with human IFNs.     

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.     

Differential induction of 2',5'-oligoadenylate synthetase by IFN-beta ser and IFN-alpha 2 in serum-supplemented and serum-free HL-60 leukemic cell cultures

The influence of cell culture conditions on the induction of 2',5'-oligoadenylate (2-5A) synthetase by recombinant interferons IFN-beta ser and IFN-alpha 2 has been investigated in human HL-60 leukemic cells. Cells maintained either in the fetal bovine serum-supplemented medium (FBS-SM) or in a serum-free, chemically defined Nutridoma-supplemented medium (SFN-SM) are treated with different concentrations of the two types of IFN and the extent of 2-5A synthetase induction compared. While cells in FBS-SM show a substantially greater increase in 2-5A synthetase by IFN-beta ser than cells in SFN-SM, the latter culture condition is significantly more effective in elevating synthetase activity with the addition of IFN-alpha 2. These data suggest that there are growth modulators and other "factors" in the fetal bovine serum which may interact specifically with each type of IFN to coordinate the optimal expression of the 2-5A synthetase protein.     

CpG oligodeoxynucleotide induction of antiviral effector molecules in sheep

Immunostimulatory CpG oligodeoxynucleotide (ODN) can protect mice against infection by many pathogens but the mechanisms mediating disease protection are not well defined. Furthermore, the mechanisms of CpG ODN induced disease protection in vivo have not been investigated in other species. We investigated the induction of antiviral effector molecules in sheep treated with a class B CpG ODN (2007). Subcutaneous injection of ODN 2007 induced a dose-dependent increase in serum levels of the antiviral effector molecule, 2'5'-A synthetase. Peak levels of enzyme were observed 4 days following ODN injection and enzyme levels remained elevated for the following 3-5 days. Repeated ODN injections induced a more sustained elevation of serum 2'5'-A synthetase activity. Finally, formulation of ODN 2007 in emulsigen increased the level of serum 2'5'-A synthetase activity and this response was CpG-specific. Elevated serum 2'5'-A synthetase activity suggested that CpG ODN acted through the induction of either interferon (IFN)-alpha or IFN-gamma. ODN 2007 did not induce detectable levels of IFN-alpha or IFN-gamma when incubated with peripheral blood mononuclear cells, but both IFN-alpha and IFN-gamma were detected following stimulation of lymph node cells with ODN 2007. CpG ODN induction of 2'5'-A synthetase in vitro correlated with the secretion of both IFN-alpha and IFN-gamma. Furthermore, immunohistochemical staining of skin revealed a marked cellular infiltration at the site of ODN 2007 injection. This cellular infiltration was CpG-specific and consisted of primarily CD172(+) myeloid cells. Many of the cells recruited to the site of ODN 2007 injection expressed IFN-alpha and some IFN-gamma. These observations support the conclusion that localized cell recruitment and activation contribute to CpG ODN induction of antiviral effector molecules, such as interferon and 2'5'-A synthetase.     

Multiple molecular forms of interferon display different specific activities in the induction of the antiviral state and 2'5' oligoadenylate synthetase

Both Hu IFN-alpha A and Hu IFN-alpha D, produced by two independent recombinant bacterial clones, are mixtures of monomers, dimers and trimers. These forms, when assayed individually in heterologous MDBK cells, induced different degree of antiviral and 2'5' oligoadenylate synthetase (2'5' A synthetase) activities: the antiviral activity of the monomer is greater than that of the dimer and the trimer, whereas the activity of 2'5' A synthetase induction is lower with the monomer than with the dimer or the trimer. Similar differences are also observed on human cells. Compared to the mononeric form, the dimeric and the trimeric forms of Hu IFN-alpha A show higher antiviral inducing activity on heterologous MDBK cells than on homologous WISH cells, whereas the 2'5' A synthetase inducing activity in these two cell lines is about the same. Thus for the same antiviral activity, the trimer or the dimer compared to the monomer are much better inducers of the 2'5' A synthetase on human than on MDBK cells.     

Interferon and growth factor modulation of nuclear factors binding to 5' upstream elements of the 2-5A synthetase gene

We assayed fragments of the 5' flanking sequence of the human 2-5A synthetase gene for their ability to respond to interferon-alpha (IFN) and platelet-derived growth factor (PDGF). Transient transfection assays identified a 40-base pair fragment, which, regardless of orientation, could confer IFN-inducibility on the thymidine kinase promoter. This same fragment was active in monkey and mouse cells and in the latter was responsive to PDGF. The effect of PDGF could be inhibited by anti-interferon antibodies. Gel retardation assays, using the 40-base pair probe, detected the presence of IFN-modulated DNA-binding factors in nuclear extracts from monkey cells. In mouse cells both IFN and PDGF induced the binding of nuclear factors to a synthetic 2-5A synthetase response sequence. Thus, both IFN and growth factors directly or indirectly modulate the binding of nuclear factors to the same region of the 2-5A synthetase gene.     

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.     

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.     

In vivo and in vitro induction of 2'-5' oligoadenylate synthetase by interferon-alpha in nodular non-Hodgkin's lymphoma and correlations with the clinical response

We investigated the correlations between the in vivo-in vitro induction of 2'-5' oligoadenylate synthetase (2-5A synthetase) by IFN-alpha in cells isolated from patients with low-grade nodular non-Hodgkin's lymphoma (NHL) and subsequent clinical responses of these patients to IFN-alpha therapy. Eleven patients were treated daily with 9 x 10(6) U of IFN-alpha 2a in a phase II trial. After an eight week treatment, four patients achieved complete remission, one a partial response, one a minor response, and five failed to respond. Basal levels of 2-5A synthetase in lymph node tumor B cells and peripheral blood mononuclear cells (PBMC) isolated before therapy differed from patient to patient and were significantly lower than in PBMC from healthy donors (P less than 0.03). In vivo single injections of 9 x 10(6) U IFN-alpha 2a induced the 2-5A synthetase in PBMC from all patients to various degrees without quantitative relation to the clinical responses. Injection of a tenfold lower dose resulted in effects of similar extent in most cases. In vitro, IFN-alpha 2a induced the 2-5A synthetase in lymph node tumor B cells isolated before therapy, and the degree of induction was significantly higher in patients who proved to respond to therapy than in patients who displayed no or minor responses (P less than 0.013). This indicates that, in nodular NHL, the 2-5A synthetase assay may have some predictive value for responsiveness to IFN-alpha therapy.     

Interferon and phorbol esters down-regulate sIgM expression by independent pathways

We studied the effects of recombinant interferon, 12-0-tetradecanoylphorbol-13-acetate (TPA), and phorbol 12, 13 dibutyrate (PDB) on surface immunoglobulin expression by Daudi cells. Incubation of cells with recombinant alpha 2 interferon (IFN-alpha 2) caused a 2.5-fold (60%) decrease in sIgM expression as measured by relative fluorescence index (RFI) using a flow cytometer. This decrease in sIgM expression was independent of inhibitory effects on proliferation and cell cycle progression. TPA or PDB also caused a threefold (67%) decrease in sIgM expression, while enhancing proliferation and cell cycle progression. Coincubation of cells with IFN-alpha 2 and TPA decreased sIgM expression by more than fourfold (greater than 75%), which was greater than the decrease induced by the optimal concentration of either agent alone. Molecular studies demonstrated that the treatment of cells with IFN-alpha 2 or TPA decreased the steady-state levels of mRNA for the heavy chain of IgM (c mu), suggesting that down-regulation of sIgM occurred at a pretranslational level. Activation of the cell membrane sodium/proton antiport did not play an integral role in the IFN-alpha 2 or phorbol-ester-induced pathway of sIgM down-regulation. Whereas IFN-alpha 2 induced an increase in the activity of 2',5'-oligoadenylate (2-5A) synthetase, the addition of TPA to IFN-alpha 2 caused a significant decrease in the activity of this enzyme. Although IFN-alpha 2 and TPA exhibited additive effects on sIgM expression, they had opposing effects on cell proliferation, cell cycle progression, and induction of 2-5A synthetase activity, suggesting that these agents down-regulate sIgM expression through independent pathways.     

Interferon induction of (2′–5′) oligoisoadenylate synthetase in diploid and trisomy 21 human fibroblasts: Relation to dosage of the interferon receptor gene (IRFC)

Trisomy 21 human fibroblasts are more sensitive to human interferon-alpha (IFN-alpha) than are diploid controls, consistent with the location of the gene (IFRC) which codes for the IFN-alpha receptor on chromosome 21. When compared in the antiviral assay, the difference in sensitivity is five- to tenfold, much greater than the 50% difference in IFRC gene dosage. An understanding of the mechanism by which this amplification of gene dosage occurs is relevant to the specific pathology of Down's syndrome and as a model system for studying the pathogenic effects of chromosomal aneuploidy. The enzyme (2'-5') oligoisoadenylate synthetase (2-5A synthetase), which is believed to be central to the interferon-induced antiviral response, is induced 50% more in trisomy 21 fibroblasts than in diploid controls. Thus the amplification in response occurs subsequent to the binding of IFN-alpha to its receptor and the triggering of the first set of intracellular events, the latter exemplified by the induction of 2-5A synthetase. Similar results were obtained with IFN-gamma, consistent with other evidence which indicates that a gene coding for a separate IFN-gamma receptor is also located on chromosome 21.     

Interleukin 2 induces interferon alpha/beta production in mouse bone marrow cells

We have previously reported that mouse bone marrow (BM) cells stimulated with alloantigen produce cytotoxic effector T-cell activity and produce interferon (IFN-)alpha/beta. In this report we show evidence suggesting that interleukin 2 (IL-2) may play a role in this IFN-alpha/beta production by alloantigen-stimulated BM cells. Alloantigen-induced IFN production by bone marrow cells was completely inhibited when cultures were supplemented with antisera to IL-2. Cell-free supernatants obtained at 2 days from cultures containing C57BL/6 BM cells and irradiated DBA/2J spleen cells were also shown to contain low levels of IL-2 activity and induced significant IFN production in fresh BM cells. Different IL-2 preparations were tested for their ability to induce IFN-alpha/beta production in mouse BM cells. Mouse BM cells cultured with recombinant human IL-2 or highly purified mouse IL-2 produced high levels of IFN-alpha/beta activity after 2-3 days of culture with significant IFN activity being detected as early as 24 hr of culture. IL-2-induced IFN-alpha/beta production was partially resistant to irradiation. In contrast, irradiated (2000 rad) bone marrow cells failed to produce any IFN when cultured with alloantigen in the absence of IL-2. T-cell-depleted BM cells or BM cells obtained from C57BL/10 nude mice produced high levels of IFN-alpha/beta following stimulation with IL-2. In addition, bone marrow cells depleted of Ia+, Qa 5+, or Asialo GM+1 cells produced IFN in response to IL-2. Thus, neither T cells nor NK cells are required for IL-2-induced IFN-alpha/beta production by BM cells. The action of IL-2 on bone marrow cells to induce IFN production was mediated by the classical IL-2 receptor, since monoclonal antibodies to the IL-2 receptor present on T cells blocked this response and since bone marrow cells depleted of IL-2 receptor-bearing cells failed to produce IFN when cultured with IL-2. These results suggest that non-T cells resident in the BM have receptors for IL-2 and can produce IFN-alpha/beta upon stimulation by IL-2. Since IFN has been shown to affect different aspects of hematopoiesis, the production of IFN by BM cells stimulated by IL-2 may be important in the control of hematopoiesis. In addition, IL-2-induced IFN production may play a role in graft-versus-host disease.     

Hyporesponsiveness to augmentation of murine natural killer cell activity in different anatomical compartments by multiple injections of various immunomodulators including recombinant interferons and interleukin 2

Augmentation of natural killer (NK) cell activity has been observed after the single administration of a wide variety of biological response modifiers (BRM); however, multiple injections of BRM have resulted in hyporesponsiveness to NK augmentation in both preclinical and clinical studies. In these studies, hyporesponsiveness to augmentation of NK cell activity occurred after multiple injections of interferon (IFN recombinant human IFN-alpha A/D and recombinant IFN-gamma) and interleukin 2 and was found to be systemic (lungs, liver, blood, and spleen). In contrast, hyporesponsiveness to augmentation by multiple injections of maleicanhydride divinyl ether (MVE-2) or Propionibacterium acnes was limited to the spleen and peripheral blood lymphocytes, with continued augmentation of NK cell activity in the peritoneum, lungs, and liver. Despite the hyporesponsiveness to augmentation of NK activity by multiple IFN injections, NK activity could still be augmented by a single injection of another BRM. The NK cell hyporesponsiveness induced in the spleen by MVE-2 was also reversed by a single administration of IFN or polyinosinic-polycytidylic and poly-L-lysine solubilized by carboxymethyl cellulose but not by OK-432 or P. acnes. These results demonstrate that the nature of the hyporesponsiveness to NK augmentation, which is induced by multiple treatments with BRM, varies with the type of agent. The noncytokine BRM that were studied induced hyporesponsiveness only in specific lymphoid compartments but not in major nonlymphoid organs, whereas cytokine BRM induced a systemic hyporesponsiveness. The hyporesponsive state induced by the different types of BRM, also varied in regard to the pattern of susceptibility to augmentation of NK activity by unrelated BRM.     

Expression of interferon-inducible genes in RD-114 cells.

RD-114 is a cell line which is partially responsive to interferon (IFN). Although both IFN-alpha and IFN gamma inhibit production of the resident retrovirus, they do not inhibit replication of other viruses, such as vesicular stomatitis virus and encephalomyocarditis virus, in these cells. In the studies reported here, we studied the characteristics of induction of seven IFN-inducible mRNAs in RD-114 cells. We observed that mRNAs 561, 6-16, 1-8, 2A, and 6-26 have similar induction characteristics in RD-114 cells and in HeLa cells, a fully responsive line. mRNA 2'-5'-oligo-adenylate synthetase (2-5(A) synthetase), however, was induced more efficiently by IFN-alpha in HeLa cells than in RD-114 cells. The same was true for the induction of metallothionein II mRNA by IFN-gamma. However, the latter mRNA was induced equally strongly in both lines when ZnCl2 was used as the inducer, suggesting that the gene is not defective in RD-114 cells. Although IFN-alpha induced 2-5(A) synthetase mRNA poorly and IFN-gamma did not induce it at all in these cells, a mixture of IFN-alpha and IFN-gamma induced this mRNA quite effectively, to a level of induction comparable to that in HeLa cells. Only 1 U of IFN-gamma per ml was sufficient to elicit this synergism, and the data suggested that an IFN-gamma-inducible protein was needed for this process. Induction of mRNA 561 by IFN-alpha in RD-114 cells, unlike that in HeLa cells, did not need ongoing protein synthesis. Once induced, this mRNA turned over rapidly in both cell lines, and this turnover could be slowed down by inhibiting protein synthesis in either cell line. IFN-induced mRNAs, such as 561 and 1-8, were polysome associated in IFN-treated RD-114 cells, suggesting that they were actively translated. Therefore, it is unlikely that the products of these IFN-inducible genes, by themselves, mediate the inhibition of replication of those viruses which are insensitive to IFN action in RD-114 cells.     

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.