Talia Hahn

ASSAY OF AN INTERFERON-INDUCED ENZYME IN WHITE BLOOD CELLS AS A DIAGNOSTIC AID IN VIRAL DISEASES

Interferon is part of the system of defence against viral infections and has important cell-regulatory and immunoregulatory functions. It is, however, not always possible to quantify circulating interferon in patients. An assay has been developed to measure an interferon-induced enzyme in white blood cells. The activity of this enzyme is constant in healthy subjects but increases by 2-10 times in 85% of patients with acute viral infections. It is also enhanced in autoimmune diseases and in virus-related malignancies and neurological disorders. The enzyme level was not raised in bacterial infections or non-infectious diseases studied. This simple and rapid biochemical assay of the interferon system could be used for diagnosis and evaluation of many diseases.

Tumour necrosis factor production and cell-mediated immunity in anorexia nervosa.

Fourteen patients with anorexia nervosa (AN) were studied for the production of tumour necrosisfactor (TNF), the activation of the interferon (IFN) system and cell‐mediated cytotoxicity (CMC)and the results were compared with 16 age‐matched healthy women. AN patients had significantlyincreased spontaneous TNF production by peripheral blood mononuclear cells (PBMC) in vitro(16 ± 5 U/ml versus 4 ± 3 U/ml in the control group; P < 005), although no TNF was detectable in theplasma from either group. TNF production in vitro, following stimulation of PBMC byphytohaemagglutinin (PHA) or tumour cells, was similar in AN patients and controls; however, lipopolysaccharide (LPS) induced TNF production was found to be lower in AN (P <0.1). CMC wassignificantly lower in AN patients (4 ± 2 versus 10 ± 3 in controls, expressed as lytic units/106 cells;P<0.05), but no difference could be found between AN and controls in IFN activity as reflected bythe level of the IFN‐induced enzyme 2′‐5′oligoadenylate synthetase (2‐5A) in PBMC. Beta‐endorphins in the plasma were higher in the AN group (P < 0.05) but these levels could not becorrelated to those of lFN, CMC or TNF. Defective CMC and increased TNF production by PBMCin patients with anorexia nervosa may possibly result from the nutritional deficiencies andneuroendocrine abnormalities associated with the disease, and may contribute to the pathophysiology of AN.

Interrelated effects of tumor necrosis factor and interleukin 1 on cell viability

Cells are sensitized to the cytolytic effect of tumor necrosis factor (TNF) by simultaneous application of inhibitors of RNA or protein synthesis. Treating cells, in the absence of such inhibitors, with cytokine preparations produced by stimulated mononuclear leukocytes may render them resistant to the cytolytic effect of TNF + the inhibitors. One of the cytokines which induces that resistance was identified as TNF itself (17). As shown in the present study, similar resistance against TNF-mediated killing can be effectively induced also with preparations of cytokines which are depleted of TNF. Fractionation of such TNF-free preparations revealed that their resistance-inducing activity is mediated by interleukin 1 (IL 1). In part of the cell lines in which IL 1 induced resistance to TNF killing, when applied without inhibitors of protein/RNA synthesis, it was found to exert cytolytic effect in the presence of such inhibitors, however, less effectively than TNF. Both TNF and IL 1 thus appear to activate in cells cytolytic mechanisms as well as antagonizing mechanisms which can protect cells from cytolysis.

Increase of vulnerability to lymphotoxin in cells infected by vesicular stomatitis virus and its further augmentation by interferon.

The cytotoxic effect of lymphotoxin (LT) and its modulation by interferon (IFN) was quantitatively assessed in uninfected and vesicular stomatitis virus (VSV)-infected cultured cells. Preparations of human LT, which were depleted of IFN, had a significant cytotoxic effect on VSV-infected HeLa, SV-80, WISH, and Vero cells. IFN, most notably IFN-gamma, further potentiated destruction of the infected cells by these LT preparations, when applied on the cells at sub-antiviral IFN concentrations. In contrast, no cytotoxic effect could be observed in any of the examined cells, when applying LT, IFN, or their combination, in the absence of viral infection. Infected cells in which VSV replication was suppressed by treatment with antiviral concentrations of IFN also resisted destruction by LT. These findings indicate that LT cytotoxicity can be selectively directed against virus-infected cells and that IFN can augment this cell-killing mechanism when failing to exert an antiviral effect.

Lymphoblastogenesis in Down's syndrome and its inhibition by human interferon

In addition to its antiviral effect, interferon has been reported to have biological effects on cell growth [ 1,2] and on the immune system [3,4]. DNA synthesis in mitogen-stimulated lymphocytes was shown to be inhibited by interferon [5-71. In human cells, a gene on chromosome 21 has been implicated in determining the antiviral response to interferon, possibly through control over the interferon receptor [8-IO]. It was not clear whether chromosome 21 also plays a role in the non-viral effects of interferon. It therefore seemed of interest to compare the sensitivity to interferon of lymphoblastogenesis in cells trisomic for chromosome 21 (as found in Down’s syndrome) as compared to normal (disomic 21) subjects. Incorporation of [3H]thymidine in lymphocytes treated with mitogens and interferon was used as an in vitro measure of cell proliferation resulting from antigen stimulation in vivo. The results reported indicate that this non-viral effect of interferon on lymphoblastogenesis is also regulated by genes on chromosome 2 1. The possible role of chromosome 2 1 in cell-cell interactions and in the immunodeficiency seen in Down’s syndrome is discussed.

Enhanced release of lymphotoxins by interferon-treated cells

Pretreatment of human peripheral blood lymphocytes with interferon significantly enhances the release of lymphotoxins (LTs) observed at subsequent incubation of the cells, for 3 hr, with phytohemagglutinin (PHA). Fractionation of the LTs by gel filtration shows that interferon (IFN) strongly increases the release of certain LTs produced by these cells, while it has little effect on the release of others. The release of LTs from the IFN-treated cells is dependent on stimulation by PHA, requires Ca2+ ions, and can be blocked by prostaglandin E1, but it is independent of protein synthesis.

Reduced production of tumor necrosis factor by mononuclear cells in hairy cell leukemia patients and improvement following interferon therapy.

In 16 patients with hairy cell leukemia (HCL) there was a marked reduction in the production of cytotoxins (CTXs) by peripheral blood mononuclear leukocytes in response to stimulation in vitro by phytohemagglutinin (PHA), 4β‐phorbol‐12‐myristate‐13‐acetate (PMA), or Sendai virus. CTX yields of 23.5 ± 21.5 U/ml, 15 ± 18 U/ml, and 12.1 ± 12.1 U/ml were obtained in response to PHA, PMA, and Sendai virus, respectively, as compared with corresponding yields of 207.3 ± 93.1, 154 ± 37.4, and 205.2 ± 62.4 in healthy controls. The extent of reduced production of CTXs appeared to be correlated with the severity of the disease. Systemic interferon (IFN) administered to four patients caused CTX production to improve in response to PHA (147.5 ± 55.1 U/ml compared with pretreatment values of 14.1 ± 6 U/ml, P < 0.05). However, CTX production in response to Sendai virus remained low. The extent to which CTX production by hairy cell leukemia mononuclear cells was reduced was proportionate to the observed decrease in monocyte counts. However, the degree to which CTX production improved after IFN treatment was significantly greater than the observed increase in monocyte counts. The major CTX induced by PHA in mononuclear cells of healthy donors and of IFN‐treated HCL patients was identified as tumor necrosis factor‐α.

In Vitro Effects of Recombinant TNF-α Binding Protein (rTBP-1) on Hematopoiesis of HIV-Infected Patients

Summary: Tumor necrosis factor‐alpha (TNF‐&agr;) is believed to contribute to the hematopoietic failure often observed in patients with AIDS. Soluble TNF receptors (sTNFR) compete for TNF‐&agr; with cell surface receptors and thus may block its activity. The effect of the p55 sTNFR (recombinant TNF‐binding protein‐1 [rTBP‐1]) on the clonogenic growth of hematopoietic progenitor cells from 27 HIV‐infected patients was evaluated in comparison with 11 normal study subjects. Peripheral bloodderived, myelopoietic (i.e., granulomonocytic colony‐forming cells [GM‐CFC]) and erythropoietic (i.e, burst‐forming unit, erythroid [BFU‐E]) colonies were grown in 10‐day semisolid cultures with increasing concentrations of rTBP‐1. Significantly, dose‐dependent increases occurred in GM‐CFC from 17 of 21 AIDS patients and 12 of 21 in BFU‐E at rTBP‐1 concentrations of 1&mgr;/ml to 25 &mgr;/ml. In contrast, rTBP‐1 failed to induce any appreciably increased colony formation in normal cell cultures. In 6 patients treated with highly active antiretroviral treatment (HAART), TBP‐1 alone did not demonstrate the in vitro hematopoiesis‐enhancing effect. This study may provide an initial step in development of therapeutic use of TBP as a TNF‐&agr; antagonist in HIV‐infected patients who do not benefit sufficiently from antiretroviral treatment, and in other conditions in which increased levels of TNF‐&agr; may contribute to hematopoietic deficiencies.

Involvement of cytotoxins in the immune response to viral infection

A human cytotoxin (CTX) with an Mr of 17,500 was purified to homogeneity from cytokine preparations by the use of a monoclonal antibody against that protein. Sendai virus and, to a lesser extent, the lectin phytohemagglutinin were found to induce effective production of that CTX in cultures of human peripheral-blood mononuclear cells, the first - by stimulating monocytes to produce the proteins, and the latter - by stimulating T cells. With both kinds of inducers, CTX production correlated to a marked increase in the cellular levels of mRNA for CTX, as quantitated by translation of that mRNA, to biologically active CTX, in microinjected Xenopus oocytes. Crude CTX preparations, as well as purified CTX, were found to be selectively cytotoxic to metabolically depressed and to virus infected target cells; they effectively killed cells which were treated with inhibitors of macromolecule synthesis, such as cycloheximide, or infected by viruses, such as VSV, but failed to exert a cytotoxic effect in the absence of such sensitizing treatments. IFN, most notably IFN-gamma, further potentiated destruction of virus-infected cells by the purified CTX, when applied on these cells at subantiviral concentrations, while uninfected cells remained resistant to CTX following treatment with IFN. Formation of the 17.5K CTX in response to viral infection and the selective cytotoxic effect of that protein on cells infected by viruses, indicate a role of CTX in the defense against viral infections.

Translation of mRNA for human lymphotoxin in microinjected Xenopus oocytes

Synthesis and secretion of biologically active human lymphotoxin (LT) can be detected in Xenopus laevis oocytes following their inoculation with poly(A+) RNA from human stimulated peripheral blood lymphocytes, but not in oocytes inoculated with RNA from unstimulated lymphocytes or from fibroblastoid cells. In size‐fractionating mRNA of stimulated lymphocytes most LT activity is found to be coded for by RNA with an approximate sedimentation value of 19 S.