Induce IFN Production Research Articles

Antibody-Dependent Induction of Type I Interferons by Poliovirus in Human Mononuclear Blood Cells Requires the Type II Fcγ Receptor (CD32)

The induction of type I interferons (IFNs) in peripheral blood mononuclear cells (PBMCs) can be triggered by viral infection or exposure to viral glycoproteins. Here we show that the IFN-α-inducing capacity of attenuated poliovirus vaccine strains is dramatically enhanced in the presence of human polyvalent immunoglobulin G (IgG). The transcription of both IFN-α and IFN-β genes was detected by RT-PCR in stimulated cells. This antibody-dependent activation of type I IFNs genes was also observed with Formalin-inactivated or UV-inactivated poliovirus, but not with empty poliovirus capsids. The ability of poliovirus–antibody complexes to induce IFN-α was specifically inhibited when PBMCs were preincubated with an excess of the Fc fragment of IgG. Monoclonal antibodies directed to FcγRII (CD32) were also inhibitory, whereas antibodies to the two other classes of Fcγ receptors, CD16 and CD64, were not. Also, aggregation of FcγRII by anti-CD32 antibodies alone failed to induce IFN-α production. Our results suggest that induction of type I interferons by poliovirus–antibody complexes depends on CD32-mediated phagocytosis of RNA-containing viral particles. As suggested by the results of an ELISPOT analysis, only a fraction of the IFN-α-producing cells are able to synthesize IFN-α in response to poliovirus–IgG complexes.

The combination of apoptotic U937 cells and lupus IgG is a potent IFN-alpha inducer.

Patients with active systemic lupus erythematosus (SLE) have signs of an ongoing IFN-alpha production, that may be of pathogenic significance in the disease. We previously showed that SLE patients have an IFN-alpha-inducing factor in blood, probably consisting of complexes containing anti-DNA Abs and immunostimulatory DNA. The DNA component could be derived from apoptotic cells, because SLE patients have been reported to have both increased apoptosis and reduced clearance of apoptotic cell material. In the present study, we therefore investigated whether apoptotic cells, together with IgG from SLE patients, could act as an IFN-alpha inducer in normal PBMC in vitro. We found that apoptotic cells of the myeloid leukemia cell line U937 as well as four other cell lines (MonoMac6, H9, Jurkat, U266) could induce IFN-alpha production in PBMC when combined with IgG from SLE patients. The IFN-alpha production by PBMC was much enhanced when PBMC were costimulated by IFN-alpha2b. The ability of IgG from different SLE patients to promote IFN-alpha induction by apoptotic U937 cells was associated with the presence of anti-ribonucleoprotein Abs, but not clearly with occurrence of anti-DNA Abs. These results suggest that apoptotic cells in the presence of autoantibodies can cause production of a clearly immunostimulatory cytokine, which is IFN-alpha. This mechanism for induction of IFN-alpha production could well be operative also in vivo, explain the IFN-alpha production seen in SLE patients, and be important in the pathogenesis of SLE.

Lactobacilli and Streptococci activate NF-kappa B and STAT signaling pathways in human macrophages.

Gram-positive bacteria induce the production of several cytokines in human leukocytes. The molecular mechanisms involved in Gram-positive bacteria-induced cytokine production have been poorly characterized. In this work we demonstrate that both nonpathogenic Lactobacillus rhamnosus GG and pathogenic Streptococcus pyogenes (group A streptococci) induce NF-kappa B and STAT DNA-binding activity in human primary macrophages as analyzed by EMSA. NF-kappa B activation was rapid and was not inhibited by a protein synthesis inhibitor cycloheximide, suggesting that these bacteria could directly activate NF-kappa B. STAT1, STAT3, and IFN regulatory factor-1 DNA binding was induced by both bacteria with delayed kinetics compared with NF-kappa B. In addition, streptococci induced the formation of IFN-alpha-specific transcription factor complex and IFN-stimulated gene factor-3 (ISGF3). STAT1 and STAT3 activation and ISGF3 complex formation were inhibited by cycloheximide or by neutralization with IFN-alpha/beta-specific Abs. Streptococci were more potent than lactobacilli in inducing STAT1, ISGF3, and IFN regulatory factor-1 DNA binding. Accordingly, only streptococci induced IFN-alpha production. The activation of the IFN-alpha signaling pathway by streptococci could play a role in the pathogenesis of these bacteria. These results indicate that extracellular Gram-positive bacteria activate transcription factors involved in cytokine signaling by two mechanisms: directly, leading to NF-kappa B activation, and indirectly via cytokines, leading to STAT activation.

Patients with Systemic Lupus Erythematosus have Reduced Numbers of Circulating Natural Interferon-α- Producing Cells

Systemic lupus erythematosus (SLE) patients often have continuous production of interferon-α (IFN-α), but production of in vitro IFN-α by peripheral blood mononuclear cells (PBMC) may be varyingly reduced. We here report that IFN-α production induced by Herpes simplex virus (HSV) in PBMC resembling immature dendritic cells and designated natural IFN-α producing cells (NIPC), was much more affected than that induced by sendai virus (SV) in monocytes. At the cell level, the frequency of HSV-activated NIPC was reduced 70-fold, but residual NIPC produced normal amounts of IFN-α (1–2U/cell). The NIPC frequency increased 10-fold in SLE-PBMC, but not in control PBMC, when co-stimulated by the combination IFN-α-γ and GM- CSF. No spontaneous IFN-α production by PBMCs was detected in SLE patients. While no SLE serum factor inhibiting IFN-α production was seen, sera of four out of 11 SLE patients induced IFN-α production in healthy control PBMC. We propose that the number of NIPC in SLE are reduced in blood because of recruitment to tissues and activation by an endogenous IFN-α inducer, as well as because of lack of co-stimulatory cytokines. IFN-α produced in SLE could be of pathogenic significance, because autoimmune diseases develop in patients with infections or tumours during IFN-α therapy.

Mapping Quantitative Trait Loci for Immune Capacity in the Pig

Immune capacity traits show considerable genetic variation in outbred populations. To identify quantitative trait loci (QTLs) for immune capacity in the pig, various measures of immune function (total and differential leukocyte counts, neutrophil phagocytosis, mitogen-induced proliferation, IL-2 production, and virus induced IFN-alpha production in whole blood cultures, and Ab responses to two Escherichia coli antigens) were determined in 200 F2 animals from a wild pig-Swedish Yorkshire intercross. The pedigree has been typed for 236 genetic markers covering all autosomes, the X chromosome and the X/Y pseudoautosomal region. Through interval mapping using a least-squares method, four QTLs with significant effects were identified; one for total leukocyte counts, one for mitogen-induced proliferation, one for prevaccination levels of Abs to E. coli Ag K88, and one for Ab response to the O149 Ag. In addition, several putative QTLs were indicated. The results from the present study conclusively show that it is possible to identify QTLs for immune capacity traits in outbred pig populations by genome analysis.

Stimulation of Natural Interferon-α/β-Producing Cells byStaphylococcus aureus

Human peripheral blood mononuclear cells (PBMCs) produced high levels of antiviral activity, as determined by bioassay, when stimulated by Staphylococcus aureus Cowan I (SAC) and E. coli. Specific immunoassays demonstrated the presence of both IFN-alpha and gamma and, for SAC, also low levels of IFN-beta. The frequencies of SAC-induced IF N-alpha-producing cells (IPCs) were up to 1-2 per 10(3) PBMCs. These IPCs expressed the HLA-DR and CD4 antigens but not CD3, CD14, or CD19, thus resembling the natural IFN-alpha-producing cells (NIPC). The SAC was more efficient as IFN inducer when heat killed than when streptomycin inhibited. The SAC was inhibitory to virally induced IFN-alpha responses, in particular when streptomycin inhibited. Both pronase treatment and mechanical disruption of SAC cells abolished their capacity to induce IFN-alpha production. Staphylococcal strains lacking or expressing low levels of protein A (SpA) showed a decreased ability to induce IFN-alpha production. However, purified SpA did not itself induce IFN-alpha. Possibly, SpA together with other bacterial surface proteins is important for the capacity of SAC to induce IFN-alpha production in NIPC.

Genetic variation in parameters reflecting immune competence of swine

Genetic variation in total and differential white blood cell (WBC) counts, phagocytic capacity of polymorphonuclear leukocytes (PMNL), virus induced interferon-α (IFN-α) production, mitogen induced proliferation and interleukin 2 (IL-2) production of mononuclear cells (MNC) in vitro was studied in blood collected from 124 Yorkshire piglets, aged 8 weeks. The piglets were the offspring from 12 sires and 31 dams. Data from an earlier experiment, including 96 piglets of seven sires and 24 dams, were added when estimating heritabilities for Con A induced proliferation and IL-2 production. The highest heritability ( h 2=0.87±0.41) was estimated for the total number of PMNL. Medium high heritabilities ( h 2=0.3−0.4) were estimated for the phagocytic capacity of PMNL, Con A induced proliferation and IL-2 production and the total number of WBC, while the heritability estimates were lower ( h 2=0.00−0.08±0.12) for the total number of lymphocytes, serum concentrations of Ig and IFN-α production. Pronounced differences between litters from various dams were found for total number of lymphocytes, IFN-α production, Con A induced proliferation and IL-2 production. The Con A induced proliferation was positively correlated ( r=0.48, P<0.001) with the IL-2 production and both these parameters were correlated ( r=0.44 and 0.37, respectively, P<0.001) to the virus induced IFN-α production. Despite these positive correlations, no parental offspring group was uniformly superior across all traits measured. However, the heritabilities estimated for the immune parameters are sufficiently high to be used as genetic markers in selection for general immune competence of swine.

Deficiency in interferon production by leukocytes from children with recurrent respiratory infections

In vitro interferon production by peripheral blood mononuclear cells from 50 children suffering from recurrent upper respiratory tract infections was examined, and compared with that of 50 healthy children. Five respiratory pathogenic viruses and Mycoplasma pneumoniae were used as inducers. Cells from every child responded to at least three out of the six inducers by interferon production. As a group, cultures prepared from patient cells showed decreased production of IFN when stimulated with adeno, rhino, corona or RS viruses or with the mycoplasma. Similar trend between the two groups of children was seen as regards influenza A virus induced IFN production in leukocyte cultures. These results corroborate our previous findings that relative deficiency in interferon production appears to be inducer-specific, and suggest that this phenomenon may have a role in the pathogenesis of recurrent respiratory infections.

Effect of Protein Kinase C Inhibitors on Interferon-beta Production by Viral and Non-viral Inducers

Induction of interferon-beta (IFN-beta) in human (BG-9), simian (CV-1) and mouse (L-929) cell lines by Sendai virus and by poly(rI). poly(rC) has been studied for its possible dependence on protein kinase C (PKC) through the use of pharmacological inhibitors (K252a and H-7) of PKC. Exposure of BG-9, CV-1 or L-929 cells to K252a (greater than or equal to 0.025 microM), a staurosporine derivative, 24 h before or after induction of IFN with poly(rI).poly(rC), inhibited by greater than 95% the production of IFN-beta. In contrast, virus-induced IFN production was enhanced threefold or more by K252a in BG-9 and L-929 but not in CV-1 cells. A naphthalene sulphonamide inhibitor of PKC, H-7, at greater than or equal to 5 microM, decreased poly(rI).poly(rC)-induced IFN production in BG-9 and CV-1 cells by 75 to 94%, but had no effect on IFN production in L-929 cells. Viral induction of IFN was not affected significantly by H-7 in BG-9, CV-1 and L-929 cells. In contrast to these results, the calmodulin inhibitor, trifluoperazine (5 to 15 microM) did not affect IFN-beta production by poly(rI).poly(rC) but significantly enhanced IFN production by Sendai virus in both human and murine cell lines. Thus, in human and simian fibroblasts the induction of IFN-beta by poly(rI).poly(rC) appears to be PKC-dependent, whereas viral induction of IFN-beta is not. Results with K252a implicate PKC in non-viral induction of IFN in mouse fibroblasts, as well. Direct measurements of PKC activity in BG-9 cells exposed to several concentrations of K252a showed that the membrane PKC activity is significantly more sensitive to inhibition by K252a than is cytosolic PKC activity. In L-929 cells, K252a inhibited membrane PKC activity similarly, but was less effective as an inhibitor of cytosolic enzyme activity than in BG-9. These studies support an integral role for PKC activity, particularly membrane-associated activity, in non-viral [poly(rI).poly(rC)] induction of IFN-beta in human, simian and mouse fibroblasts.

Production of interferon gamma by lymphocytes exposed to antibody-coated spermatozoa: a mechanism for sperm antibody production in females

The ability of sperm to induce lymphocytes from female donors to produce interferon gamma (IFN) was measured. Of nine samples where immunoglobulin was bound to ejaculated motile sperm, five (55.6%) induced IFN production. In contrast, only 1 of 35 (2.9%) sperm samples lacking bound immunoglobulins induced IFN (P less than 0.005). Incubation of donor sperm with sperm antibody containing sera from nine male patients led, in seven cases (77.8%), to acquisition by sperm of the ability to induce IFN. Lymphocytes from eight of eight females tested all responded to sperm with bound antibodies. IFN induction of Ia antigen expression on macrophage may be required for recognition of processed sperm antigens by the female's T helper cells.

The regulation of γ-interferon production by interleukins 1 and 2

Purified interleukins 1 and 2 (IL-1 and IL-2) were used to investigate their role in the production of γ-interferon (γ-IFN). Macrophage depletion from human peripheral blood mononuclear leukocytes (PBML) inhibited γ-IFN production. Addition of purified IL-1 partially restored IFN production of macrophage-depleted PBML induced by three T cell mitogens (phytohemagglutinin, PHA; concanavalin A, con A; and staphylococcal enterotoxin A, SEA), but had no effect on induction of IFN production by undepleted PBML. Therefore endogenous IL-1 production by macrophages is probably one of the mechanisms by which they act as accessory cells for IFN production by lymphocytes. A monoclonal antibody 9.6 which binds to the sheep erythrocyte (E) receptor found on human T cells inhibited IFN production. Addition of IL-2, but not IL-1, was found to reverse this inhibition. Prostaglandin E2, a macrophage product, inhibited γ-IFN production induced by PHA, Con A, and OKT3 but usually not SEA. This inhibitory effect was reversible by the addition of IL-2 but not IL-1. In the absence of mitogen IL-1 alone rarely induced any IFN production, although some IFN was produced by PBML from a small minority of donors. Without mitogen IL-2 induced IFN production only at very high concentrations and the added presence of IL-1 did not enhance this induction.

Interleukin 2 induces interferon [formula omitted] production in mouse bone marrow cells

We have previously reported that mouse bone marrow (BM) cells stimulated with alloantigen produce cytotoxic effector T-cell activity and produce interferon ( IFN-) α β . In this report we show evidence suggesting that interleukin 2 (IL-2) may play a role in this IFN- α β production by alloantigen-stimulated BM cells. Alloantigen-induced IFN production by bone marrow cells was completely inhibited when cultures were supplemented with antisera to IL-2. Cell-free supernatants obtained at 2 days from cultures containing CS7BL 6 BM cells and irradiated DBA 2J spleen cells were also shown to contain low levels of IL-2 activity and induced significant IFN production in fresh BM cells. Different IL-2 preparations were tested for their ability to induce IFN- α β production in mouse BM cells. Mouse BM cells cultured with recombinant human IL-2 or highly purified mouse IL-2 produced high levels of IFN- α β activity after 2–3 days of culture with significant IFN activity being detected as early as 24 hr of culture. IL-2-induced IFN- α β production was partially resistant to irradiation. In contrast, irradiated (2000 rad) bone marrow cells failed to produce any IFN when cultured with alloantigen in the absence of IL-2. T-cell-depleted BM cells or BM cells obtained from C57BL 10 nude mice produced high levels of IFN- α β following stimulation with IL-2. In addition, bone marrow cells depleted of Ia +, Qa 5 +, or Asialo GM 1 + cells produced IFN in response to IL-2. Thus, neither T cells nor NK cells are required for IL-2-induced IFN- α β production by BM cells. The action of IL-2 on bone marrow cells to induce IFN production was mediated by the classical IL-2 receptor, since monoclonal antibodies to the IL-2 receptor present on T cells blocked this response and since bone marrow cells depleted of IL-2 receptor-bearing cells failed to produce IFN when cultured with IL-2. These results suggest that non-T cells resident in the BM have receptors for IL-2 and can produce IFN- α β upon stimulation by IL-2. Since IFN has been shown to affect different aspects of hematopoiesis, the production of IFN by BM cells stimulated by IL-2 may be important in the control of hematopoiesis. In addition, IL-2-induced IFN production may play a role in graft-versus-host disease.

1127 INTERFERON (IFN) PRODUCTION BY MONOCYTE/MACROPHAGES IN RESPONSE TO RESPIRATORY SYNCYTIAL VIRUS (RSV) AND PARAINFLUENZA TYPE 3 (PI3)

We have previously reported on RSV and PI3 growth in adherent human peripheral blood mononuclear leukocytes (M's). (Ped Res 18(4), 279A, 1984) with both viruses younger M's (dl post-harvest) are poorer producers of new virus than older M's (d4s7) by ≥ 10 fold. In these experiments we investigated IFN production by M's in response to stimulation with these agents. M's were infected on dl, 2, 4s7 after harvest at a MOI of 0.5-0.6 and washed after 2 hrs of viral adsorption and every 24 hrs. The supernatants were monitored for: antigen production by ELISA; infectious progeny on HEP-2 monolayers; and IFN activity in a VSV plaque-reduction assay. IFN was not detected in 2hr specimens and was maximal at 24hrs after viral exposure. With PI3 IFN production inversely correlated with new virus production. The dl M's produced the highest IFN titers (800 IU) and the least virus (2×104PFU/ml) while the d4 and d7 M's produced lower amounts of IFN (100 and 0 IU) and higher viral titers (3.2-3.4×105PFU/ml). With RSV infection the dl, 2&4 M's produced minimal IFN (<25IU); the d7 M's produced 400 IU. The significance of this IFN production in the d7 M's is uncertain but did not correlate with virus or antigen production, which are lowest on dl and highest on d7. Mock infected cells produced no detectable IFN. The observed IFN activity was successfully blocked with anti-IFN antibody. These findings correlate with the clinical findings that RSV is a poor inducer of IFN while PI can induce IFN production in nasal secretions.