Altered synthesis of interferon-γ and expression of interferon-γ receptor by peripheral blood mononuclear cells from patients with IgA nephropathy and non-IgA proliferative glomerulonephritis

Previously we reported disease-specific interaction between interferon- (IFN-) and interleukin-4 (IL-4) in patients with IgA nephropathy (IgAN), suggesting the existence of unusual T cell behavior in this disease. In the present study, we investigated characteristic synthesis of interferon- (IFN-) and expression of IFN- receptor (IFN-R) in the peripheral blood mononuclear cells (PBMC) from patients with IgAN and other chronic proliferative glomerulonephritis (PGN). Heparinized peripheral blood samples were obtained from 38 patients with chronic mesangial proliferative glomerulonephritis (CGN; including 24 with IgA nephropathy) and 20 healthy controls. PBMC were isolated by gradient centrifugation and fragments were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum (FCS) for 72 hr. IFN- concentrations in supernatants were evaluated by the enzyme-linked immunosorbent assay (ELISA). Other parts of PBMC pellets were reacted with anti-human IFN-R monoclonal antibody and FITC-labeled anti-mouse second antibody for analysis of IFN-R expression on these cells by FACScan. The remaining PBMC were fractionated into CD4⁺ T cells, CD8⁺ T cells, B cells, NK, cells and macrophages using the MACS cell sorting system. The isolated cells were evaluated for IFN- or IFN-R mRNA expression by the semiquantitative RT-PCR method.In vitro IFN- synthesis was enhanced in patients with CGN, and NK cells were revealed to be responsible for such enhancement. On the other hand, the expression of IFN-R on macrophages was suppressed in CGN patients. These results suggest that impairment of regulation of the IFN- system might be involved in the development of CGN.     

Interferon-γ for treatment of severe atopic eczema in two children

Two 4- and 5-year-old children suffering from refractory atopic dermatitis were treated with recombinant interferon- (rIFN-). rIFN- was injected at 50 g subcutaneously three times a week in the first child for 3 weeks, followed by three times 25 g in week 4. In the other child two treatment courses of 4 weeks were given after a break of 2 weeks. Therapy was well tolerated. In child one reductions in eczematous body surface and severity of lesions were observed, while no beneficial effect was seen in the other. Clinical chemistry data remained unchanged. Immunological studies performed in parallel showed a decrease in total serum IgE of 50% in child 1, a decrease in spontaneous in vitro IgE production, an increase in in vitro production of interleukin-6, and a normalization of previously decreased in vitro lymphocyte responses to several mitogens. While marked immunological changes were noted during IFN- treatment, clinical benefits were not encouraging. Diminished IFN- production has been claimed to be a major pathogenic factor in atopic eczema. Our results indicate that the pathogenesis is more complex. Clinically, we were unable to confirm previous observations in adults. Further studies are needed before IFN- can be recommended for therapy of pediatric atopic eczema.Abbreviations IFN- interferon- - IL interleukin     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

Antibody response inPlasmodium vinckei malaria after treatment with chloroquine and adjuvant interferon-γ

The antibody response of mice infected withPlasmodium vinckei after treatment with chloroquine either alone or in combination with interferon- (IFN-) was determined. Sequential serum samples were drawn from BALB/c mice receiving either 240 g chloroquine on the day of infection or 120 g chloroquine plus 10⁴ units IFN- daily for 11 days beginning on day 3 prior to infection. Mice treated with additional IFN- showed an early induction of IgG2a response and a reduction in IgG1 antibodies as detected by the immunofluorescence technique at between 10 and 16 days after infection as compared with mice treated with chloroquine alone. Thus, IFN- may partly exert its antimalarial activity via the induction of IgG2a antibody formation. At 4–6 weeks after infection, when mice from both groups resisted homologous re-infection, the predominant antibody isotypes found in both groups were IgG1 and IgG2a. Serum samples obtained from mice in both treatment groups at 6 weeks after infection were used for serum transfer experiments. When parasitised erythrocytes were preincubated with such immune serum, a retardation of the course of parasitaemia by 2 days was observed.     

Suppression by interferon-γ of tumor cell-induced increase in mesothelial permeability

The effect of interferon- (IFN-) on the interaction between tumor cells and mesothelial cell layers was studied from the aspect of changes in mesothelial permeability. Mesothelial permeability was assessed as the percentage diffusion of radiolabeled albumin across the mesothelial cell sheets on Matrigel-coated filter cup assemblies. When lined gastric carcinoma cells (KATO-III) were seeded on the confluent mesothelial cell layers, the fine cobblestone appearance of the cell sheet was disrupted and mesothelial permeability significantly increased. The increase in permeability was suppressed by the addition of as little as 1 U/ml of IFN-. The effect of IFN- was observed when either the conditioned medium of tumor cells alone or the IFN--resistant tumor cells, K-562, was placed onto the mesothelium. The cobblestone appearance of the cell sheet was relatively well preserved in the presence of IFN-. In contrast, IFN- did not suppress tumor-induced mesothelial permeability. These results suggest that IFN- has the potential to protect the human mesothelial cell layers against tumor cells.     

Cytokine mRNA in the Joints and Draining Lymph Nodes of Rats with Adjuvant Arthritis and Effects of Cyclosporin A

TNF- and IL-1 promote leukocyte recruitment to arthritic joints and may contribute to cartilage degradation while regulatory cytokines such as IL-4 and IL-1RA may in part determine the course of arthritis. Here we report the pattern of TNF-, IL-1, IL-6, IFN-, IL-1RA, and IL-4 mRNA expression, detected by RT/PCR, in the talar joint and draining popliteal lymph node (PLN) of rats with adjuvant arthritis (AA). Levels of TNF- and IFN- mRNA were increased in the PLN before clinical signs of arthritis. This was followed by increases in IL-1 and IL-1RA mRNA at d9 and IL-6 mRNA at d12. PLN IL-1RA mRNA levels were positively correlated with those of IL-1 and TNF- throughout d5-d20. IL-4 mRNA levels were highest on days 7 and 20. In the synovium, a small increase in TNF-, IL-1, and IL-6 mRNA was detected on d5 then again on d12. Maximal synovial TNF- levels were reached on d20, while IL-1 peak expression was on d16 and IL-6 on d14. IL-4, IL-1RA, and IFN- mRNA was undetectable in the synovium. Cyclosporin treatment for 4 days, initiated at the height of arthritis, rapidly decreased clinical disease, and decreased migration of neutrophils and T lymphocytes into the joints. Yet no significant effect of CyA was observed on inflammatory cytokine expression, although the correlation between PLN IL-1RA and IL-1 or TNF- was lost in treated animals. Thus there is a variable pattern of cytokine gene expression in rat AA, the undetectable IL-4 and IFN- mRNA in synovium being analogous to human rheumatoid arthritis.     

Interferons in enteroviral heart disease: modulation of cytokine expression and antiviral activity

Interferon (IFN)- has a more than 120-fold higher antiviral activity than the closely related IFN- in human myocardial fibroblasts infected with the cardiotropic enterovirus coxsackievirus B3 (CVB3). CVB3 replication induces interleukin (IL)-6 and IL-8 expression in myocardial fibroblasts, and suppresses the expression of monocyte chemoattractant protein-1 (MCP-1). We investigated whether the higher antiviral activity of IFN- compared to IFN- was a result of a suppression of IL-8 expression by IFN- since previous studies had indicated that IL-8 stimulates enterovirus replication. Human myocardial fibroblasts were treated with either IFN-, IFN- or IFN- (0, 10, 100, or 1,000 IU/ml) and the concentrations of IL-6, IL-8 and MCP-1 were measured in culture supernatants by immunoassays. Both IFN- and IFN- reduced IL-6 and IL-8 expression significantly. In addition, neutralization of IL-8 in culture supernatants of myocardial fibroblasts using a monoclonal antibody demonstrated a significant reduction of CVB3 titers. Antiproliferative effects of all three IFNs were very low (<30% with 1,000 IU/ml), indicating that the suppression IL-6 and IL-8 was not related to cytotoxicity. MCP-1 expression was increased only by high concentrations of IFN- (1,000 IU/ml). By contrast, IFN- had no significant effect on IL-6, IL-8 and MCP-1 expression. In conclusion, suppression of IL-8 expression is an "immuno-modulating" feature of IFN- in human myocardial fibroblasts, which is similar to the activity of IFN-. This feature of IFN- contributes to its high antiviral activity against CVB3 and may be useful in the treatment of enteroviral heart disease.     

Flow Cytometric Analysis of In Vitro Proinflammatory Cytokine Secretion in Peripheral Blood from Multiple Sclerosis Patients

The cytokines, interferon- (IFN-), tumor necrosis factor- (TNF-rpar;, and interleukin-2 (IL-2) are important endogenous proinflammatory proteins and have been linked to disease activity in multiple sclerosis. In this study, we use flow cytometric methodology to compare the secretion of IFN-, IL-2, and TNF- from peripheral blood-derived T cells of multiple sclerosis patients to the secretion in healthy controls. The percentages of IFN-, IL-2, and TNF- secreting cells are not significantly different between multiple sclerosis patients and controls. However, the TNF- secreting CDS cell percentage is correlated with the IFN- and IL-2 secreting CD3 cell percentages in multiple sclerosis patients. In the controls, only the TNF- secreting CD3 cell percentage is correlated with IFN-. These findings show that correlated secretion of cytokines occurs in multiple sclerosis and suggest that concerted intercytokine interactions may play an important role in the disease.     

Transient Exposure of Human Bronchial Epithelial Cells to Cytokines Leads to Persistent Increased Expression of ICAM-1

Effects of several cytokines on kinetics of Intercellular Adhesion Molecule-1 (ICAM-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1) expression were studied on a bronchial epithelial cell line (BEAS-2B). VCAM-1 was neither constitutively expressed on BEAS-2B cells nor induced by Interferon-gamma (IFN-), Tumor Necrosis Factor-alpha (TNF-), Interleukin-1beta (IL-1), IFN-, IL-4, IL-6, IL-8 or Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF). ICAM-1 was constitutively expressed on BEAS-2B cells. IFN- and TNF- upregulated ICAM-1 expression on these cells. The functional importance of IFN- plus TNF- upregulation of ICAM-1 expression on BEAS-2B cells was demonstrated by neutrophil-BEAS-2B cell adhesion assays. Cytokines are rapidly released and cleared in animals. Therefore, transient cytokine(s) exposure might occur on the bronchial mucosa. Brief incubation of BEAS-2B cells with IFN- plus TNF- led initial upregulation of ICAM-1 expression followed by a protracted downregulation. Our findings stress the importance of studying the mechanism(s) controlling the persistent increased expression of ICAM-1 after brief cytokine(s) exposure.     

Differential secretion of TNF-α and IFN-γ by human peripheral blood-derived NK subsets and association with functional maturation

Natural killer cells can be separated into three major subsets (free, binder, and killer) based on their ability to bind and kill sensitive target cells. The nonbinder, nonkiller free cells are the most immature and can be activated to become binders and killers. Natural killer (NK) cells synthesize and secrete several cytokines that are intimately involved in NK activation. This study investigated the secretion of tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) by purified NK cells and NK subsets following activation by various stimuli. K562 target cells stimulated secretion of both TNF- and IFN- by both the binder and the killer subsets but not by the free subset. IFN- activated the secretion of IFN- only, whereas IL-2 activated the secretion of both TNF- and IFN- by the binder and killer subsets and secretion was augmented by the addition of K562 to the cultures. Phorbol myristate acetate (PMA) and ionophore stimulated TNF- and IFN- secretion in both the binder and the killer subsets, though IFN- secretion was more pronounced in the binder subset. Activation of TNF- and IFN- secretion was dependent on de novo protein synthesis. Analysis at the single-cell level demonstrated that the binder subset had the highest frequency of cells secreting IFN-. These results demonstrate that both the binder and the killer subsets can be activated to secrete TNF- and IFN-, whereas the free NK subset secretes little or no TNF- and IFN- following activation. These data suggest that the ability of NK cells to secrete TNF- and IFN- following activation correlates with the functional stage of maturation of NK cells.     

Interaction of G-actin with thymosin β4 and its variants thymosin β9 and thymosin β 9 met

Summary Thymosin ₄ is a major actin sequestering peptide in vertebrate cells and plays a role in the regulation of actin monomer/polymer ratio. Thymosin ₉ and thymosin ₉ ^met are minor variants of thymosin ₄. The possible function of these peptides has been investigated by comparing the actin binding properties of these -thymosins. Thymosin ₉ and thymosin ₉ ^met were found to inhibit polymerization of ATP-actin with identical K _d s of 0.7–0.8 M (as compared to 2±0.3 M for thymosin ₄); like thymosin ₄, they bound to ADP-G-actin with a 100-fold lower affinity than to ATP-G-actin. The interaction of thymosin ₄ and thymosin ₉ ^met with G-actin was weakened 20-fold upon oxidation of methionine-6 into methionine sulfoxide. Binding of thymosin ₄ to G-actin was accompanied by a 15% increase in the fluorescence intensity of actin tryptophans, and a 10 nm emission blue shift. Methionine-6 played an important role in this effect. The fluorescence change was used to monitor the kinetics of thymosin ₄ binding to G-actin in the stopped-flow. The reaction was bimolecular, with association and dissociation rate constants of 1.5 M^-1 s^-1 and 2s^-1 respectively, under physiological conditions. The possible physiological significances of methionine-6 oxidation and of the relatively slow binding kinetics in regulating thymosin ₄ function in vivo is discussed.     

Interferon cures cells lytically and persistently infected with African swine fever virus in vitro

Summary Human interferon alpha (IFN-) and interferon gamma (IFN-) inhibit African swine fever (ASF) virus replication in Vero cells. IFN- and IFN- exert a synergistic inhibition. Human tumor necrosis factor (TNF) does not inhibit ASF virus replication in this cell line, but in combination with IFNs it has antiviral enhancing activity. Analysis of the mechanism of inhibition suggests that the action of these cytokines blocks a step that comes prior to DNA replication. The 2-5 A synthetase activity is induced in Vero cells by treatment with these cytokines and is activated after ASF virus infection. More interesting is the finding that continuous treatment with IFN- cures Vero cells from lytic and persistent infections with ASF virus. A potential application of IFN for the treatment of animals carrying the virus is suggested.Part of this work was presented at the VII International Poxvirus-Iridovirus Meeting, Heidelberg, Federal Republic of Germany, August 22–26, 1988.     

Characterization of the hog cholera virus 5′ terminus

Hog cholera virus (HoCV) 5 terminus of the ALD and GPE(–) strains were analyzed by using rapid amplification of cDNA end method (5RACE). An additional nine nucleotides were found at the 5 termini of genomic RNA in the ALD and GPE(–) strains of HoCV. These nine nucleotides were also conserved in BVDV and were suggested to form a hairpin structure at the 5 terminus by computer-assisted analysis. It seems possible that the secondary structure and/or the 5 terminus sequence has a significant role in the HoCV virus genome.     

Interferon β1a Treatment Modulates TH1 Expression in γδ + T Cells from Relapsing-Remitting Multiple Sclerosis Patients

A paradigm exists that multiple sclerosis is causally related to dysregulation of T_H1 inflammatory cytokines and T_H2 antiinflammatory cytokines. The cytokine source(s) that initiate the imbalances are unknown. In this study, , CD4, and CD8 T cell receptor-positive (TCR+) cells were isolated from the blood of 26 definitive relapsing-remitting multiple sclerosis patients prior to interferon -1a (IFN1a) therapy and following 8–10 weeks of this therapy. The bioactivities of interferon (IFN), interleukin 10 (IL10), and interleukin 12 (IL12) were determined. The concentrations of IFN, IL10, and IL12 from each cell type did not change significantly with IFN1a treatment. The IL10 secreted by TCR+ cells strongly correlated with the IL12 secreted by the same TCR+ cells, supporting the paradigm. Furthermore, IFN1a therapy decreased the TCR+ cell secretion of T_H1 cytokines after 8–10 weeks of therapy.     

Sublocalization on chromosome 21 of human interferon-alpha receptor gene and the gene for an interferon-gamma response protein

The cellular responses to alpha and beta interferons (IFN- and -) are mediated through the IFN-/ (type I) receptor, while the response to IFN- is mediated through the IFN- (type II) receptor. The receptors for IFN-/ and IFN- are encoded by genes on human chromosomes 21 and 6q, respectively. The presence of chromosome 21q confers both ligand binding and responsiveness to human IFN-/, whereas chromosome 6q confers binding of Hu-IFN-, but not cellular responsiveness on somatic cell hybrids. Chromosome 6q (i.e., the Hu-IFN- receptor gene) and chromosome 21q are both necessary for the cellular response of somatic cell hybrids (from fibroblasts) to Hu-IFN-. It is conceivable that the factor mediating activity through the IFN- receptor is, in fact, the IFN- receptor, or that the two genes are distinct but part of an interferon response region. Here we more precisely localize on human chromosome 21 the genes for the IFN- receptor and for the factor(s) mediating the action of IFN- through the chromosome 6-encoded receptor. Hamster-human somatic cell hybrids containing various fragments of human chromosome 21 were used. The presence of the human IFN-/ receptor was determined by binding³²P-labeled human IFN- to cells, covalently cross-linking the [³²P]IFN--receptor complex, and analyzing it by SDS-polyacrylamide gel electrophoresis. The presence of the IFN- receptor-related factor mediating cellular responsiveness was determined by HLA induction in hybrid cells containing the IFN- receptor (chromosome 6q), a transfected copy of the human HLA-B7 gene, and various portions of chromosome 21. In all hybrids examined, the two genes cosegregate. Specifically, both genes are localized to the region of chromosome 21 containing the markers D21S58, D21S65, and GART and appear to be proximal to D21S58. The implications for IFN action are discussed.     

Effect of rat andβ-human interferons on hyperacute experimental allergic encephalomyelitis in rats

Human interferon- (human IFN-) and rat interferon (rat IFN) were evaluated on experimental allergic encephalomyelitis (EAE) in rats, a delayed cellular reaction resembling human multiple sclerosis (MS). Rat IFN was active by intravenous and intracerebroventricular routes. It decreased the severity of clinical symptoms of paralysis during the 22 days of the assay. Human IFN-, on the contrary, had no effect when similarly tested in this rat model. Cyclophosphamide delayed the onset of paralysis, but levamisole enhanced the severity of the EAE.     

Cytokine profile in systemic lupus erythematosus,rheumatoid arthritis,and other rheumatic diseases

We investigated serum levels of interleukin-6 (IL-6), interferon-gamma (IFN-), and tumor necrosis factor alpha (TNF) from patients with systemic lupus erythematosus (SLE) and its various clinical manifestations of disease and from patients with rheumatoid arthritis (RA) and other rheumatic diseases. The serum levels of IL-6 and IFN- were highly elevated from patients with SLE associated with lymphadenopathy (LN) or nephrotic syndrome (NS). On the contrary, the serum levels of TNF were elevated from most patients with SLE associated with thrombocytopenia (TP). However, serum levels of TNF were in the normal range from patients with SLE associated with NS, LN, or central nervous system disease. Of interest, patients with SLE associated with humoral immunodeficiency disorder, hypogammaglobulinemia, had highly elevated levels of serum IL-6. The concanavalin A-stimulated mononuclear cells (MNC) of patients with SLE associated with TP secreted highly elevated levels of TNF compared to other patient groups. We suggest that abnormal production of various cytokines in SLE is an intrinsic defect of MNC and the immune system that may be the key element for a variety of clinical manifestations of this disease.     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.