Enhancement of T cell cytotoxic responses by purified human fibroblast interferon.

Purified polyribonucleotide-induced human fibroblast interferon (HFIF) was tested for its effects on proliferative and cytotoxic human T cell responses to alloantigens. The addition of HFIF (100 to 400 IFU/ml) to mixed leukocyte cultures decreased alloantigen-induced lymphocyte proliferative responses as determined by both recovery of responding cells and by 3H-thymidine incorporation into responding cells. However, HFIF, but not the mock interferon preparation, increased the cytotoxic response of T cells to allogeneic cells by 4- to 5-fold when expressed in terms of lytic units. Although fibroblast and leukocyte interferons have different physicochemical and biologic properties, the results reported here are in concert with previous findings concerning the effects of virus-induced leukocyte interferon on human T cell functions.     

Human interferon enhances gunaylate cyclase activity

Partially purified human leukocyte interferons, partially purified human lymphoblastoid interferon, and human fibroblast interferon enhanced rat liver, kidney, and splenic guanylate cyclase {E.C.4.6.1.2.} activity 2–4 fold at 5 μIU concentration. Dose-response relationships revealed that the human leukocyte interferons enhanced splenic guanylate cyclase activity at concentrations as low as 0.01 μIU while a concentration of 1 μIU for partially purified human lymphoblastoid interferon and 10 μIU concentration for human fibroblast interferon were necessary to see any effect on guanylate cyclase activity.     

Enzymatic modifications of human fibroblast and leukocyte interferons

The sensitivity of highly purified human fibroblast interferon and partially purified human leukocyte interferon to several proteolytic and glycolytic enzymes was determined with respect to antiviral activity, isoelectric point, molecular weight, and thermal stability. Leucine aminopeptidase altered the distribution of isoelectric points for both interferons but produced little change in molecular weights; this enzyme somewhat reduced the activity of only leukocyte interferon. Treatment of fibroblast interferon with carboxypeptidases A and B did not greatly decrease antiviral activity, but it did slightly reduce the molecular weight of the interferon and substantially altered the distribution of isoelectric point values; similar treatment of leukocyte interferon caused some loss in activity, especially of the 17,000-molecular-weight species. Both interferons were inactivated rapidly by treatment with the endoproteases trypsin, pepsin, bromelain, and subtilisin. Chymotrypsin shifted the isoelectric points of both interferons, but only leukocyte interferon was significantly inactivated. Treatment with neuraminidase and beta-galactosidase changed the isoelectric point distribution but did not affect the activity or thermal stability of either interferon; such a treatment reduced the molecular weight of fibroblast interferon and the size heterogeneity of leukocyte interferon. Treatment with neuraminidase and then leucine aminopeptidase greatly reduced the activity of both interferons, especially leukocyte interferon. The data indicate that biologically active forms of fibroblast and leukocyte interferons can be distinguished by their relative sensitivity to certain proteases.     

Bacteria-derived human leukocyte interferons alter in vitro humoral and cellular immune responses

Cultures of gradient-purified human peripheral blood mononuclear cells (PBMC) have been employed to examine the effects of three bacteria-derived human leukocyte interferon subtypes on certain aspects of in vitro immune responses. The addition of highly purified IFN-alpha 1, -alpha 2, -alpha 2/alpha 1 to PMBC cultures stimulated with phytohemagglutinin (PHA) or pokeweed mitogen resulted in a significant suppression of the mitogenic response. This suppression required the presence of interferon in the cultures because pretreatment of cells and removal of interferon had no effect on their response to PHA. The presence of these interferons at 200 U/ml also caused a substantial reduction of human mixed-lymphocyte reactions (MLR) as measured by [3H]thymidine incorporation by responder cells. Interestingly, pretreatment of stimulator cells was sufficient for this reduction to occur whereas pretreatment of responder cells had no effect on their ability to respond to allogenic stimulation. In contrast to these suppressive effects, the three interferons enhanced human in vitro primary immune response to sheep red blood cells (SRBC). These data demonstrate that both purified interferon subtypes and genetic hybrids of human interferons produced by recombinant DNA technology have effects on in vitro immune responses.     

Mitogen dose-dependent effect of weak pulsed electromagnetic field on lymphocyte blastogenesis

The effects of pulsed extremely low frequency electromagnetic fields on human peripheral blood lymphocyte mitogenesis induced by phytohaemoagglutinin, concanavalin A or calcium ionophore A23187 were studied. The dependence of the field effect on mitogen concentrations was investigated. Field exposure produced strong inhibition of DNA synthesis when optimal doses of mitogens were used, confirming our previous findings. Opposite effects were observed at suboptimal concentration of mitogens. Experiments performed by exposing cell cultures to the field for short periods indicated that a field application of at least 6 h is needed to influence irreversibly lymphocyte blastogenesis.     

The effect of natural and recombinant leukocyte interferons on DNA repair and the formation of chromosome aberrations induced by N-methyl-N'-nitro-N-nitrosoguanidine

The anticlastogenic action of natural leukocyte and recombinant (alpha 2) interferons was studied in human lymphocyte cultures treated with N-methyl-N'-nitro-N-nitrosoguanidine. The criteria of cell viability, proliferation, chromosome aberrations, frequency of micronucleus formation, formation and repair of DNA breaks were used for estimation of interferons activity. Reduction of the induced chromosomal aberrations was obtained in cells pretreated with interferons. The protective effect of natural leukocytic interferon was more expressed as compared with the effect of recombinant (alpha 2) interferon. The natural interferon was also more efficient than the recombinant one in DNA breaks formation and repair.     

Dissociation of protein kinase activity and the induction of the antiviral state in a cell line responsive to the antiviral effects of interferon

Interferons or oxidized glutathione were found to induce double-stranded RNA-dependent protein kinase activity in mouse L cells that phosphorylates the α subunit of eukaryotic peptide initiation factor 2. A mixture of leukocyte/fibroblast interferons as well as immune interferon induced the protein kinase and also suppressed virus replication in the L cells. Oxidized glutathione was equally effective in inducing protein kinase activity, but it did not induce an antiviral state in these cells. The data suggest that a simple cause and effect relationship does not exist between protein kinase induction and the establishment of the antiviral state in a cell that is responsive to the antiviral effects of interferon.     

Human leukocyte interferon (HuIFN-alpha): potent endorphin-like opioid activity

Human leukocyte interferon, but not fibroblast or immune interferons, binds to opiate receptor $\text{in}\text{,}\text{vitro}$. When injected intracerebrally into mice, human leukocyte, but not fibroblast or immune interferon, caused potent endorphin-like opioid effects. These effects include analgesia, lack of spontaneous locomotion and catalepsy. All of these actions of human leukocyte interferon were preventable and reversible by the opiate antagonist naloxone. The findings suggest that some of the side effects of leukocyte interferon therapy may be mediated by opiate receptor binding. They also provide evidence for a regulatory circuit between the immune and neuroendocrine system. This putative circuit could be an etiologic site for certain psychopathological states.     

Antiviral and protein-inducing activities of recombinant human leukocyte interferons and their hybrids.

The antiviral activities of recombinant human leukocyte interferons IFN-alpha A and IFN-alpha D as well as five hybrids of these interferons against retroviruses, vesicular stomatitis virus, and encephalomyocarditis virus were studied in feline, human, and murine cells. Although these interferon species had widely different potencies, their activities against these viruses were, in general, proportional. The IFN-alpha A/D (Bgl) hybrid was the most potent species, and the IFN-alpha D/A (Bgl) hybrid was the least potent. However, the latter species did not interfere with the action of the former species. Like natural human leukocyte interferon, each of the seven species of recombinant interferons induced the synthesis of at least five proteins in human fibroblasts, whereas induction of only one such protein was readily detected in a feline fibroblast line in which these interferon species inhibited the replication of all three viruses.     

Molecular structure of human fibroblast and leukocyte interferons: probe by lectin and hydrophobic chromatography.

Structural differences between human leukocyte virus-induced interferon and human fibroblast polyinosinic-polycytidylic acid (rIn-rCn)-induced interferon have been noted in previous studies. This study reports the behavior of human leukocyte and fibroblast interferon, induced by virus and by rIn-rCn, in several lectin and hydrophobic chromatographic systems. Differences in both glycosylation and in hydrophobicity of human leukocyte and fibroblast interferons are documented. Human fibroblast interferon is a glycoprotein, whereas our evidence suggests that human leukocyte interferon probably is not. Also, fibroblast interferon is more hydrophobic than leukocyte interferon, as probed on several hydrophobic adsorbents. The possible relationships of these differences to each other and to antigenic variations are discussed. Generally, the differences appear to be attributable to the cell type in which the interferon was induced. However, our results suggest that at least subtle differences in the processing of the induction signal (virus or rIn-rCn) within the same cell type may occur, slightly altering some structural features.     

Human leukocyte interferon has no structural or biological relationship to corticotropin

In view of recent reports that certain preparations of human leukocyte interferons are structurally and biologically related to the pituitary hormones corticotropin (ACTH) and β-endorphin, we have investigated the properties of two human leukocyte interferons (IFN-α) prepared by recombinant DNA technology. The antiviral activities of purified IFN-αA and IFN-αD were not affected by a large molar excess of ACTH antiserum nor did ACTH interfere in interferon immunoassays. Neither IFN-αA, IFN-αD nor pepsin digests of these proteins were able to stimulate steroidogenesis in adrenocortical cells. There was no cross reaction between ACTH antiserum and the two leukocyte interferons or the pepsin digests of the interferons. These results cast doubt on recent proposals that some of interferon's biological effects are mediated by ACTH or β-endorphin-related fragments of the interferon molecule.     

Apparent dispensability of the carbohydrate moiety of human interferon for antiviral activity.

Human leukocyte and tritium-labeled fibroblast interferons, prepared by induction with Sendai virus and with double-stranded polyinosinic acid.polycytidylic acid respectively, have been studied in relation to the carbohydrate moieties attached to them. These interferons were partially purified by immunoabsorbance and by gel filtration. On treatment with glycosidases, about 80% of the 3H-labeled sugar moieties in this glycoprotein-containing fraction was removed without detectable alteration of the antiviral activity or antibody-binding properties characteristic of interferon. The molecular weight of leukocyte interferon was reduced by about 4000. As others have reported, the heterogeneous character of interferon revealed by isoelectric focusing was greatly reduced by the enzyme treatment.     

Classification of interferons with antibody to immune interferon

Mouse immune Interferon, induced by the T-cell mitogen staphylococcal enterotoxin A (SEA), was partially purified and used to immunize rabbits. The resulting antiserum neutralized all immune interferon preparations tested, including interferon induced in vitro by SEA, concanavalin A, phytohemagglutinin P, and pokeweed mitogen, and in mixed lymphocyte cultures. Interferon produced in vivo with specific antigen was also neutralized. The antiserum was equally potent against all these interferon preparations. The serum did not neutralize any virus-type interferon preparation tested, but immune interferon induced by SEA in athymic nude mouse spleen cells was neutralized. The neutralizing activity was precipitable by 33% ammonium sulfate, and was not removed by absorption of the serum with mouse cells. The data suggest that immune interferons produced under diverse conditions are antigenically the same or closely related.     

Effect of interferons on dengue virus multiplication in cultured monocytes/macrophages

The effects of interferons on dengue virus multiplication in cultured human and mouse monocytes/macrophages were studied. Interferon treatment before, but not after virus inoculation suppressed virus multiplication dose-dependently. Recombinant human leukocyte A interferon was as effective as ordinary human fibroblast interferon in suppressing dengue virus multiplication in cultured human monocytes. Human monocytes, a population of non-proliferating cell lineage, maintained their interferon-mediated antiviral state for a few days after removal of the interferons.     

Presence of human chromosome 21 alone is sufficient for hybrid cell sensitivity to human interferon.

Human/mouse somatic cell hybrids with chromosome 21 as the only detectable human genetic material were sensitive to both human leukocyte and fibroblast interferons. The presence of additional human chromosomes decreased the amount of interferon needed to attain a given level of virus resistance. Decreased cytopathic effects, decreased virus yields, and the appearance of a specific phosphorylated protein associated with interferon treatment were all observed in hybrids maintaining only human chromosome 21. The phosphorylated protein found in extracts of these human interferon-treated hybrid cells was of mouse origin.     

Interferon: pharmacokinetics and toxicity

Interferons disappear rapidly from the serum of animals and man, and the kidney may be the major site of interferon destruction. The relevance of serum levels of interferons to their therapeutic activity has not been clearly established, particularly as the stimulation of host defence mechanisms by interferons may be important. Relatively low serum levels of antiviral activity are seen after intramuscular injections of fibroblast interferon compared with those after the same dose of leucocyte interferon. Injections of very pure leucocyte and lymphoblastoid interferons from several sources cause fever, headaches, malaise and myalgia associated with a corticosteroid response and probably with inflammatory prostaglandin synthesis. These reactions become less with repeated dosing but very large doses of lymphoblastoid interferon have been shown to cause liver damage and serious metabolic disturbances. Treatment with moderate doses of exogenous interferons may occasionally be associated with the development of neutralizing antibodies.     

Four sulfhydryl-modifying compounds cause different structural damage but similar functional damage in murine lymphocytes

Four thiol-modifying compounds were used to inhibit murine lymphocyte mitogenesis. The compounds were a copper sulfate/O-phenanthroline complex (CuP) to oxidize surface thiols, N-ethyl maleimide (NEM) to alkylate surface and intracellular thiols, D,L-buthionine-S,R-sulfoximine (BSO) to prevent synthesis of glutathione, and hydrogen peroxide, which reacts with various cellular constituents, including sulfhydryls. Splenic lymphocytes were incubated with one of the four compounds, washed, and then stimulated with the B cell mitogen, LPS, or the T cell mitogen, Con A. In spite of their differing chemical reactivities and differing effects on cell viability, lipids, and total, protein, and non-protein thiols, the four sulfhydryl-modifying compounds had very similar effects on the kinetics and inhibition of lymphocyte growth. All compounds had complex effects on mitogenesis, causing enhanced, delayed, or inhibited tritiated thymidine incorporation. Although the total thiol contents of untreated T cells and B cells were found to be equivalent, the LPS response consistently was inhibited by lower concentrations than the Con A response, suggesting that B cells were more sensitive than T cells to thiol modification. To compare compounds the efficiency of inhibition was determined by functionally relating reductions in mitogenesis with reductions in thiol content of the cells. The compounds differed in inhibitory efficiency; thus, damage to some thiols must be more important than damage to others. CuP ablated mitogenesis with the least change in thiol content. Therefore, surface sulfhydryls appear critical in lymphocyte mitogenesis. With all compounds inhibition of mitogenesis occurred over a very narrow range of thiol content, suggesting that the thiols important in inhibition were few in number relative to the total thiol content of the cell.     

Molecular weight of the functional unit of human leukocyte, fibroblast, and immune interferons

There is good agreement between the target molecular weight and the known molecular weight of human leukocyte interferons (about 20,000). The target molecular weight of fibroblast interferon, 31,000 to 42,000, is significantly larger than the monomer molecular weight of 21,000 to 24,000, suggesting that the dimer may be the predominant active functional unit in solution. A range from 63,000 to 73,000 for the target molecular weight of several different fractions of immune interferon (including natural crude as well as the recombinant form) indicates that the functional form of the immune interferon may be a trimer or tetramer. Thus, these studies indicate that the functional unit of leukocyte interferon is the monomer, that of fibroblast interferon is a dimer, and that of immune interferon is probably a tetramer (or trimer).     

Reversible inhibition by interferon of the maturation of human peripheral blood monocytes to macrophages

We demonstrated that interferon delays the maturation of human monocytes to macrophages in vitro as assessed by morphologic, histochemical, and biochemical parameters. After exposure to interferon, monocytes were slightly smaller, less stretched out, and had a delayed loss of granules with peroxidase positive reactivity, as compared with control, noninterferon-treated cells. Also, interferon prevented the increase of the specific activity of three lysosomal enzymes (β-galactosidase, β-glucuronidase, and β-N-acetylglucosaminidase) in the monocytes, and enzyme activities were 30–40% of that observed in untreated cells. Both human leukocyte and human fibroblast interferons were effective in delaying the maturation process. The effects of the interferon were species specific and reversible and were neutralized by antiinterferon serum. These studies describe a new nonantiviral effect of interferon, unique in that it involves the delay of maturation of cells in a system which is not associated with cell proliferation. Thus interferon could potentially effect host defense mechanisms and aspects of the immune response which are dependent on the maturation of monocytes to macrophages.     

Interferon treatment reduced adherence, invasiveness and intracellular multiplication of Shigella flexneri in coxsackie B1 virus-infected cells.

The effect of interferon treatment on interaction of Shigella flexneri with in vitro cultured cells was investigated. Pretreatment of HEp-2 cells with human interferons had no effect on the susceptibility of cells to S. flexneri, measured by invasiveness and adhesiveness. Human leukocyte interferon and human recombinant interferon-alpha-A reduced adhesiveness, intracellular multiplication and invasiveness of S. flexneri in HEp-2 cells preinfected with coxsackie B1 virus. Also non-receptor mediated-phagocytosis was reduced by interferon treatment in virus infected cells. The interferon effects were dependent on continuous protein synthesis, because they were not expressed when cycloheximide or abrin was added to the virus infected cell cultures. No effect of interferon was detected on intracellular content of Na+ or K+, Na(+)-K+ activated ATPase activity or cytoplasma membrane polarity, in virus infected or control cell cultures. The interferon effect on bacterial invasiveness seems to be dependent on an interferon receptor interaction on cytoplasma membrane level because directly microinjected interferon showed no effect.