Amichai Schattner

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.

Review: Interferons and Autoimmunity

ABSTRACT Autoimmunity can be accelerated in several genetically prone murine models and can even be induced in normal mice by treatment with interferon (IFN) or IFN-inducers. Several cases of IFN-induced autoimmune disease in humans also have been observed; however, more striking is the fact that some of the clinical manifestations in autoimmune diseases and many of the immunological aberrations can be mediated or enhanced by IFN. The finding of high levels of circulating IFN in many patients may be highly significant in that respect, and the characterization of the predominant type of IFN as an unusual acid-labile IFN-α may indicate an infectious etiologic agent in autoimmunity, since this peculiar IFN was mostly associated with viral infections in vivo or in vitro. The induction of MHC class II antigens on previously HLA-DR or la negative cells appears to be caused primarily by IFN-γ and may have a central role in the pathogenesis of autoimmunity in susceptible individuals. Such aberrant HLA-DR expression on nonlymphoid cells can be detected early in the disease in the target organs of many varied autoimmune conditions and may trigger a cascade of self-directed, uncontrolled immune response in conjunction with other factors.

Involvement of interferon in virus-induced lymphopenia

Intraperitoneal injection of vesicular stomatitis virus (VSV) into mice causes marked and rapid changes in leukocyte distribution. The virus induces an increase in peripheral blood (PB) granulocytes and an extensive decrease in the lymphocyte count which reaches a nadir of less than 10% of preinfection values, 12 hr after virus inoculation. In the lymph nodes and spleen extensive lymphocyte translocation and granulocyte infiltration are observed. Most changes abate 48 hr following virus inoculation. Injection of poly(rI):(rC) causes similar changes to those observed with VSV. The lymphocyte changes observed after injection of VSV or poly(rI):(rC) coincide with high levels of interferon (IFN) in the serum. We have examined the effects of anti-IFN antibody on those changes and investigated whether they can be mimicked by injecting IFN. Our findings suggest that the IFN induced by VSV or poly(rI):(rC), rather than those agents themselves, causes the observed lymphopenia as well as some of the changes observed in the spleen. On the other hand, the effects of VSV on granulocyte localization do not appear to be mediated by IFN.

The origin of autoantibodies

Autoimmune diseases are characterized by the appearance of autoantibodies (autoAb) which may participate in their pathogenesis, but autoAb have also been found in normals with a variety of other conditions. The production of hybridomas from lymphocytes of unimmunized normal mice and healthy humans and analysis of the monoclonal autoAb (m-autoAb) obtained, showed that many had polyspecific autoAb reactivity, binding to many seemingly unrelated self-antigens, or to several organs. Most m-autoAb were of the IgM class and shared a common cross-reactive idiotype (CRI). Low levels of Ab with similar binding pattern and idiotype are continuously represented in the serum of mice and humans who have no evidence of autoimmune or other disease. Very similar Ab appear in autoimmune diseases. Studies of m-autoAb derived from lupus-prone mice and from patients with systemic lupus erythematosus (SLE) and other autoimmune diseases also revealed polyspecific binding, IgM isotype and CRI. Moreover, these CRI, which were almost identical with the idiotypes of natural autoAb in normals, may identify a group of pathogenic Ab in the lupus mice and SLE patients. Since the data clearly suggest that lymphocytes that make autoAb are common and are part of the normal B cell repertoire coded by widely dispersed germline genes, there remain the basic problems of the function of these autoAb in health, as well as the question of their regulation and activation in vivo. Several postulated functions and immunoregulatory mechanisms are discussed and the possible role of certain factors, especially viruses, in enhancing autoAb production and autoimmunity, is assessed.

INCREASED (2'-5')-OLIGO-A SYNTHETASE ACTIVITY IN PATIENTS WITH PROLONGED ILLNESS ASSOCIATED WITH SEROLOGICAL EVIDENCE OF PERSISTENT EPSTEIN-BARR VIRUS INFECTION

initial finding of raised anti-VCA IgM antibody. Very high levels of enzyme activity, similar to those observed in acute infectious mononucleosis,’ were found in all the patients tested (see table). Interferons (IFNs) are produced in response to viral infections. Among the biochemical changes caused in cells by IFNs is the induction of the enzyme oligo-A synthetase,2 thus an increased activity of this enzyme indicates that the host cells have been exposed to IFN and are responding to it. Increased levels of this enzyme have been observed during acute viral infections, collagen diseases, and EBV-related malignancies. In mice injected intraperitoneally with vesicular stomatitis virus and Sindbis virus, (2’-5’)oligo A levels return to normal within 1-4 weeks.3 After acute infectious mononucleosis, activity of the enzyme seems to decline to normal levels over several weeks. Where the illness was prolonged, increased levels of enzyme activity were found many months after the onset of signs and symptoms. Enzyme activity was measured in these patients when there was no evidence of an acute viral infection or of malignant or any other disease associated with increased synthetase activity. In our Jan. 9 paper we suggested that there may be some defect in the IFN system in these patients. The data presented here do not necessarily rule this out. The similarity to the response in acute viral infections supports the existence of continuing viral infection in this group of patients.

Lysis by natural killer cells requires viral replication and glycoprotein expression

Several lines of evidence suggest that natural killer cells (NK) have an important role in antiviral defense. To be thus effective, NK cells have to recognize and cause lysis of virus-infected cells. The mechanism underlying this interaction was investigated in a murine system using vesicular stomatitis virus (VSV). A large number of cell lines was screened to identify a permissive target for VSV infection and the B16 murine melanoma cells were chosen since VSV replicates in these cells without producing cytopathic effects 24 h after infection. Spontaneous lysis of B16 melanoma cells by spleen cells occurred only if the target cells were previously infected with VSV. Treatment of spleen cells with anti NK 1.1 or anti Thy 1.2 plus complement decreased the specific lysis by 50%, therefore, the phenotype of the effector cells in this system corresponds to the NK cell subset. Immunofluorescent staining with polyclonal and monoclonal anti-VSV antibodies demonstrated that the viral glycoprotein G is abundantly expressed on the target cell surface. Treatment of the VSV-infected targets with tunicamycin prevented glycosylation of newly synthesized VSV glycoprotein G on the cell membrane. This treatment abrogated completely NK-mediated killing of the infected B16 melanoma cells. These results indicate that virus replication and membrane expression of glycosylated protein G are essential for recognition and lysis of VSV-infected targets by NK cells.

Differences in susceptibility to NK cell killing of two cloned sublines derived from a single clone

The ability of natural killer (NK) cells to discriminate between virus infected or tumor cells and their normal counterparts indicates a highly selective recognition, but the exact target structure remains unknown. We have examined two clones of measles virus persistently infected HeLa cells derived from the same parental clone, one of which, HeLa-mss, is highly susceptible to NK killing, (35% specific lysis at 80:1 lymphocyte to target ratio), whilst the other, HeLa-msr, is totally resistant (0.4-2.4% specific lysis). The HeLa-msr cells also failed to inhibit lysis of the HeLa-mss target cells, indicating that HeLa-msr cells did not share the receptor of HeLa-mss for NK cells. The expression of the two measles virus-encoded surface antigens, the hemagglutinin (HA) and fusion (F) proteins on the plasma membrane was examined by immunofluorescent staining and FACS analysis using monoclonal antibodies to the respective proteins. Approximately equivalent amounts of HA and F antigens were found on the cells of the HeLa-msr and HeLa-mss clones. Immunoprecipitation and PAGE analysis also failed to reveal any qualitative differences in the migration patterns or relative proportion of the measles virus proteins between these cell lines. Nevertheless, the in vitro differences in susceptibility of the two cell lines to NK lysis were found to be important in vivo, since HeLa-mss cells failed to grow as tumors in nude mice while HeLa-msr were highly tumorigenic. Thus, NK cells may either recognize very subtle differences in viral encoded polypeptides, possibly point mutations, or alternatively, they may recognize changes in the host cell membrane induced by insertion of certain viral encoded polypeptides.

INTERFERON-INDUCED PROTEINS: BIOLOGICAL FUNCTIONS AND CLINICAL APPLICATIONS

ABSTRACT The induction by Interferons of the synthesis of specific cellular proteins can be applied to investigate whether cells have been exposed to IFNs. For both the (2′-5′) oligo A synthetase and the HLA cell surface antigens, induction by IFNs was demonstrated at the level of mRNA, and the specificity for various IFNs was studied. Clinical applications of the synthetase assay in peripheral white blood cells are described.