Enzymatic induction of interferon production by galactose oxidase treatment of human lymphoid cells.

Human lymphocyte cultures produced large amounts of interferon after treatment with the enzyme galactose oxidase. Interferon production was detectable as early as 3 h after enzymatic treatment and reached a level of about 10(4) reference units 20 to 24 h later. Galactose oxidase-induced interferon appeared to be immune interferon on the basis of acid lability, lack of neutralization by antibody to leukocyte interferon, and slow kinetics of activation of the cellular antiviral state. Interferon production was inhibited to the same extent (99%) by pretreatment of the cells with beta-galactosidase or with neuraminidase followed by beta-galactosidase, suggesting that the critical event for activation of interferon production is the oxidation of exposed galactose residues on lymphocyte membrane.     

Immune interferon induced by phytohemagglutinin in nude mouse spleen cells.

Phytohemagglutinin is able to trigger interferon synthesis in spleen cell cultures from nude (nu/nu) mice as effectively as in splenic cell cultures from haired, control (nu/+), thymus-bearing mice. A minor theta-bearing cell population present in the spleen of nude mice appears essential to phytohemagglutinin interferon production, although cooperating cells are also required. The properties of nude mouse phytohemagglutinin interferon are indistinguishable from those displayed by the interferon induced in thymus-bearing mouse spleen cell cultures. Both interferons are unstable at pH 2 and cannot be neutralized by an antiviral interferon serum; hence, their characteristics correspond to those described for type T interferon. As in the case of viral interferon, pretreatment of L cells with nude phytohemagglutinin interferon induced specific enhanced phosphorylation of a 67,000-molecular-weight protein in vitro when cell extracts were incubated with double-stranded RNA and gamma-[32P]ATP.     

Human immune interferon: induction in lymphoid cells by a calcium ionophore

Human lymphocyte cultures produced interferon after treatment with the calcium ionophore A23187, but not after treatment with potassium ionophore nonactin. Interferon production was detectable between 3 and 6 h after ionophore treatment and reached the maximum level between 12 and 24 h. Ionophore-induced interferon appeared to be immune interferon on the basis of acid lability, lack of neutralization by antibody to leukocyte interferon, slow kinetics of activation of the antiviral state, and molecular characterization by isoelectrofocusing.     

Immune regulation by non‐lymphoid cells in transplantation

Regulatory cells play a crucial role in the induction and maintenance of tolerance by controlling T cell as well as B and natural killer (NK) cell‐mediated immunity. In transplantation, CD4⁺CD25⁺forkhead box P3⁺ T regulatory cells are instrumental in the maintenance of immunological tolerance, as are several other T cell subsets such as NK T cells, double negative CD3⁺ T cells, γδ T cells, interleukin‐10‐producing regulatory type 1 cells, transforming growth factor‐β‐producing T helper type 3 cells and CD8⁺CD28^‐ cells. However, not only T cells have immunosuppressive properties, as it is becoming increasingly clear that both T and non‐T regulatory cells co‐operate and form a network of cellular interactions controlling immune responses. Non‐T regulatory cells include tolerogenic dendritic cells, plasmacytoid dendritic cells, mesenchymal stem cells, different types of stem cells, various types of alternatively activated macrophages and myeloid‐derived suppressor cells. Here, we review the mechanism of action of these non‐lymphoid regulatory cells as they relate to the induction or maintenance of tolerance in organ transplantation.     

The role of suppressor cells in the production of macrophage migration inhibition factor.

The production of migration inhibition factor (MIF) by human peripheral blood lymphocytes and guinea pig lymph node lymphocytes, in response to mitogen, concanavalin A, or antigen, tuberculin, has been studied. Suppressor cells have been depleted by in vitro ageing of cultures for 24 hours, and this has resulted in a significant increase in MIF activity. Reconstitution of the aged lymphocyte population by the addition of fresh lymphocytes results in a suppression of MIF production. When lymphocytes, which have been cultured for 24 hours in the presence of antigen or mitogen to generate suppressor cells, are added to aged cultures MIF production is inhibited. These results suggest that suppressor cells play a regulatory role in the production of MIF.     

Non-genotype-specific role of the hepatitis C virus 5' untranslated region in virus production and in inhibition by interferon

The 5′ untranslated region (5′UTR) of hepatitis C virus (HCV) is structured into four domains (I-IV) with numerous genotype-specific nucleotides. It is unknown whether the polymorphisms confer genotype-specific functions to the 5′UTR. Using viable JFH1-based Core-NS2 recombinants, we developed and characterized HCV genotypes 1-7 recombinants with highly diverse 5′UTRs (genotypes 1a and 3a), 2a recombinants (J6/JFH1) with 5′UTR of genotypes 1-6 or with heterotypic chimeric (1a/3a and 3a/1a) 5′UTR domains I, II or III, and 1a recombinants with 5′UTR domain I of genotypes 1-6. All were fully functional in Huh7.5 cells; therefore, the 5′UTR apparently functions in a non-genotype-specific manner in HCV production in vitro. However, adenine at the 5′-terminus was required. We demonstrated that J6/JFH1 with 5′UTR of genotypes 1-6 responded similarly to interferon-α2b. This study provides novel insight into the role of the 5′UTR in the HCV life cycle and facilitates HCV basic research and testing of 5′UTR-targeting antivirals.     

Suppression of interferon production in mouse spleen cells by cytochalasin D.

Cytochalasin D is thought to impair microfilament function. The present study has investigated its effects on four different systems in which interferon is formed, namely (1) mouse fibroblasts induced with virus (2) mouse spleen cells induced with virus, or (3) with endotoxin or (4) by allogeneic stimulation. Cytochalasin D did not suppress formation of interferon by fibroblasts (L cells) or spleen cells stimulated with either HVJ or NDV. However it did suppress production of interferon by spleen cells in response to endotoxin or an allogeneic stimulation; here its action was apparently not on the secretion of interferon, but on some earlier event. It also suppressed the production of interferon by mouse spleen cells induced with HVJ if this had been u.v. irradiated for more than 15 min: this suggests that cytochalasin D sensitive structures do play some role in interferon production by mouse spleen cells when stimulated with HVJ, as well as when they are stimulated with endotoxin or an allogeneic stimulus.     

Antibody production by transferred lymphoid cells: Inhibition by competition of antigens

A/Jax and C57BL mice were immunized with either human red blood cells or with both human cells and (A/Jax×C57BL)F₁ hybrid spleen cells, and sensitized spleen cells were transferred to isogeneic or hybrid hosts. Anti-human haemagglutinin titres were lower in hosts receiving C57BL rather than A/Jax cells, were lower in hybrid than isogeneic hosts, and were lower in animals receiving doubly- than in those receiving singly-sensitized cells.

The results can be best interpreted on the basis of competition of antigens, rather than by postulating allergic death of sensitized lymphoid cells exposed to an overwhelming dose of antigen.

Competition of antigen was found to occur in A/Jax and C57BL mice actively immunized with both human red cells and hybrid spleen cells, when antibody titres were compared with those of singly-immunized controls. Anti-human haemagglutinin titres in actively immunized hybrid mice were intermediate between those of the parental strains, suggesting that differences in titres between the parental strains were due to physiological differences in the antibody-forming system, rather than to the more poorly reacting strain sharing antigenic determinants with and therefore being partially tolerant to human red cells.

     

Inhibition of human herpesviruses by combination of acyclovir and human leukocyte interferon.

Human leukocytes interferon in low concentrations (1 to 5 U/ml) enhanced the antiviral effect of acyclovir against herpes simplex virus, varicella-zoster virus, and cytomegalovirus grown in human fibroblasts. This occurred without additive inhibition of the division of human fibroblasts or proliferation of peripheral blood mononuclear cells. The combined antiviral effect was additive against clinical isolates of cytomegalovirus and was synergistic against clinical isolates of the other two viruses. The magnitude of the effect with cytomegalovirus was the same when laboratory and wild-type virus were compared. The persistence of varicella-zoster virus in the presence of acyclovir in infected human cells was also reduced by the addition of interferon.     

Immune interferon. II. Different cellular site for the production of murine macrophage migration inhibitory factor and interferon.

The production of macrophage migration inhibitory factor (MIF) and immune interferon (IF) by concanavalin A (Con A)-stimulated cultures of thymus, lymph node and spleen cells was investigated. It was found that all cultures produced MIF activity, whereas only spleen cells produced marked IF activity. The capacity to produce IF was found to be correlated with the macrophage content of a cell preparation as evidenced by staining for esterase-positive cells. Furthermore, column-purified spleen T cells produced MIF but no IF. Migration inhibition caused by residual mitogen could be ruled out. On the other hand, when macrophages grown from bone marrow cells were pre-exposed to supernatants of mitogen-stimulated lymphocytes, IF activity was released into freshly added medium while no significant MIF activity was found. IF was also found in supernatants of macrophage cultures after exposure to conventional inducers in vitro (polyinosinic-polycytidylic acid, Corynebacterium parvum) or in vivo (C. parvum), whereas no MIF was detected. An anti-Type I IF serum neutralized IF in supernatants from Con A-stimulated spleen cells but did not affect MIF in the same supernatants. This indicates that IF and MIF activity are associated with different molecules. It is, therefore, concluded that under the described conditions, IF and MIF are produced by different cells. T cells are the prime producers of MIF while IF is released by macrophages following induction by lymphokines.     

The role of the lymphoid organs in antibody production

Summary Simultaneous removal of the thymus gland and spleen in rabbits does not affect the viability of these animals. Together with the other organs the thymus gland and the spleentake part in the response of the animal to the administration of the typhoid antigen. This participation is manifested in various forms: thymectomy promotes greater accumulation of the antibodies in the blood; splenectomy on the contrary reduces the titre: Simultaneous removal of the thymus and spleen results in a greater accumulation of antibodies than in the case of splenectomized animals, although it is slightly less than in the normal ones.Thus one may assume that the thymus has a depressing effect on the mechanisms responsible for the accumulation of the antibodies in the blood; this effect is especially clearly manifested in the absence of the spleen. This is also confirmed experimentally by the increased titer of circulating antibodies in splenectomized rabbits after thymectomy, as well as by experiments with administration of thymus homogenate. The depressive effect of the thymus gland is manifested only during the first 48 hours after the administration of the antigen.From the Division of Infectious Pathology and Experimental Therapy of Infections (Head-Corresponding Member AMN SSSR Kh. Kh. Planel'es) of the N. F. Gamaleya Institute of Epidemiology and Microbiology (Director-Prof. S. N. Muromtsev)Presented by Active Member AMN SSSR L. A. Zil'ber     

Regulation of the interferon system: evidence that Vero cells have a genetic defect in interferon production.

A clone of Vero cells was isolated and shown to be totally unable to synthesize interferon and insensitive to the toxic effect of poly(rI).poly(rC) treatment. Cells of this clone and mouse L cells were fused by treatment with polyethylene glycol or Sendai virus. Hybrid cell clones were isolated following selection in medium containing hypoxanthine, thymidine and ouabain. The hybrids were sensitive to the antiviral effect of poly(rI).poly(rC) and synthesized mouse, but not primate, interferon. It is proposed that in Vero cells, the gene for interferon synthesis is defective or absent.     

Enhancement of the expression of histocompatibility antigens of mouse lymphoid cells by interferon in vitro

The expression of surface antigens of thymocytes and splenic lymphocytes was determined by quantitative absorption of alloantibodies. Mouse interferon preparations enhanced the expression of histocompatibility antigens of thymocytes from mice of different ages, but of splenic lymphocytes only from young mice. Interferon did not affect the expression of the theta antigen of thymocytes or splenic lymphocytes.     

Replication of herpesviruses in human cells transformed by cytomegalovirus.

The replication of human cytomegalovirus (CMV) and herpes simplex virus (HSV) was studied in three human embryo cell lines (CMV-Mj-HEL-I, CMV-Mj-HEL-2, and CMV-Mj-HEL-2,T-I) transformed in vitro by human CMV. Growth studies revealed that these cells were completely resistant to infection by CMV strains ADI69 and Mj and partially resistant to HSV types I and 2. Neither virus DNA nor virus proteins were synthesized in the transformed cells infected with CMV AD169. The HSV production in CMV-transformed human embryo lung (HEL) cells was delayed when compared to the virus production in normal HEL cells and spread of HSV c.p.e. was slower in the transformed cells. The treatment of normal HEL cells with a crude extract of CMV-transformed HEL cells also resulted in inhibition of the spread of c.p.e. of HSV types I and 2. The inhibitory effect was not due to interferon since vesicular stomatitis virus replication was not affected and several experiments showed that it was not due to mycoplasma. The presence of virus inhibitor molecules in CMV-transformed cells absent in normal HEL cells is postulated.     

Spontaneous interferon production and Epstein-Barr virus antigen expression in human lymphoid cell lines.

Established human lymphoid cell lines, many of which spontaneously produce interferon, differ in the efficiency by which they allow expression of Epstein-Barr virus (EBV) lytic functions. Six EBV carrying lymphoid cell lines, selected to either be extremely susceptible or very refractory to EBV superinfection, were tested for spontaneous interferon production. Only the three cell lines which were poorly superinfectable with EBV were found to produce interferon. These same three lines could not be induced to express EBV-specific early antigens from intrinsic EBV genomes. It is suggested that interferon acts as a negative control factor affecting a cell's susceptibility to EBV.     

Factors influencing the production of interferon from L cells.

The production of interferon from Newcastle disease virus-infected mouse L929 cells was investigated in relation to superinduction procedures, cell density, cellular cyclic adenosine 3',5'-monophosphate (cAMP) levels, and rate of incorporation of 14C-labeled protein hydrolysate into trichloroacetic acid-precipitable material. Densely populated cultures did not have their interferon production enhanced through "superinduction" using cycloheximide, actinomycin D, or the two antimetabolites in combination. These dense cultures produced more interferon per cell than less dense cultures, even though the interferon production from the latter cells could be enhanced two- to threefold by cycloheximide or combined cycloheximide and actinomycin D. Cells in densely populated cultures relative to those from sparsely populated cultures were smaller in volume, had a correspondingly reduced protein content and a lower concentration of cAMP, and were less able to concentrate 14C-labeled protein hydrolysate, although proportionally they were just as efficient in incorporating labeled precursors into trichloroacetic acid-precipitable polypeptides.     

Disparity in the production of lymphoblastogenesis inhibition factor by cultured human B and T lymphoid cell lines.

A comparative study of inhibitory effects of cell-free supernatants from cultured human B and T lymphoid cell lines on lymphocyte blastogenesis indicated that the inhibitory effect of supernatant from B lymphoid cells on lymphocyte blastogenesis was significantly higher than that of supernatant from T lymphoid cells or from non-lymphoid neoplastic cells. The inhibitory effect of supernatant was reversible and dose-related. The inhibitory effect gradually diminished with time when the supernatant from B lymphoid cells was added to the culture, 1-3 days after the beginning of cultures. The supernatant of human B lymphoid cells was also found to be highly active in affecting the mouse thymus cell response. The biological nature of this inhibitory factor has not been defined. Both B lymphoid cell lines used in the present study contained Epstein-Barr virus (EBV) genomes while the T-cell line and the non-lymphoid neoplastic cell lines were free of EBV genomes. Sensitivity of the supernatant of B lymphoid cells to u.v. irradiation and heat suggests the possibility that the EBV genomes released into the culture medium may be responsible for inhibition of lymphocyte blastogenesis; resistance of this supernatant to DNase suggests that the EBV genomes may be double-stranded DNA.