Npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type I interferon induction at local replication sites

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The viral protein Npro of CSFV interferes with alpha- and beta-interferon (IFN-α/β) induction by promoting the degradation of interferon regulatory factor 3 (IRF3). During the establishment of the live attenuated CSF vaccine strain GPE-, Npro acquired a mutation that abolished its capacity to bind and degrade IRF3, rendering it unable to prevent IFN-α/β induction. In a previous study, we showed that the GPE- vaccine virus became pathogenic after forced serial passages in pigs, which was attributed to the amino acid substitutions T830A in the viral proteins E2 and V2475A and A2563V in NS4B. Interestingly, during the re-adaptation of the GPE- vaccine virus in pigs, the IRF3-degrading function of Npro was not recovered. Therefore, we examined whether restoring the ability of Npro to block IFN-α/β induction of both the avirulent and moderately virulent GPE--derived virus would enhance pathogenicity in pigs. Viruses carrying the N136D substitution in Npro regained the ability to degrade IRF3 and suppress IFN-α/β induction in vitro. In pigs, functional Npro significantly reduced the local IFN-α mRNA expression in lymphoid organs while it increased quantities of IFN-α/β in the circulation, and enhanced pathogenicity of the moderately virulent virus. In conclusion, the present study demonstrates that functional Npro influences the innate immune response at local sites of virus replication in pigs and contributes to pathogenicity of CSFV in synergy with viral replication.

Defective antitumor function of monocyte -derived macrophages from epithelial ovarian cancer patients

An abundance of monocytes and macrophages (MO/MA) in the microenvironment of epithelial ovarian cancer (EOC) suggests possible dual roles for these cells. Certain MO/MA subpopulations may inhibit tumor growth by antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or stimulation of adaptive immunity. In contrast, other MO/MA subpopulations may support tumor growth by immunosuppressive or pro-angiogenic cytokine production. A better understanding of the phenotype and activity of MO/MA in EOC should lead to greater insight into their role in the immunopathobiology of EOC and hence suggest targets for treatment. We have found differences in the proportions of MO/MA subpopulations in the peripheral blood and ascites of EOC patients compared to normal donors, and differences in MO/MA surface phenotype in the associated tumor environment compared to the systemic circulation. We also demonstrate that, following their activation in vitro, monocyte-derived macrophages (MDM) from the peripheral blood and ascites of EOC patients exhibit antitumor effector activities that are different from the behavior of normal donor cells. The phenotypic characteristics and antitumor activity of CD14+ MO/MA and an isolated subpopulation of CD14brightCD16 −HLA-DR+ MO/MA were compared in samples of normal donor peripheral blood and the peripheral blood and ascites from EOC patients. MDM were cultured with macrophage colony-stimulating factor (M-CSF) and activated with lipopolysaccharide (LPS) or a combination of LPS plus recombinant interferon-gamma. We determined that MO/MA from EOC patients had altered morphology and significantly less ADCC and phagocytic activity than did MO/MA from normal donors. ADCC and phagocytosis are mediated by receptors for the Fe portion of IgG (FcγRs), the expression of which were also found to be deficient on EOC MDM from peripheral blood and ascites. Anti-tumor functions not mediated by the FcγRs, such as macrophage mediated cytotoxicity and cytostasis, were not impaired in EOC MDM compared to normal donor MDM. Our findings also showed that MDM from both EOC patients and normal donors produce M-CSF-stimulated cytokines, including interleukin-8, tumor necrosis factor alpha, and interleukin-6, which have the potential to support ovarian tumor growth and metastasis. These findings may be relevant to the pathogenesis of EOC and to the development of future bioimmunotherapeutic strategies. ^

Percentage of cultured cell-populations that stained positively and/or negatively for apoptotic markers cleaved caspase-3 and cleaved PARP, following DNA damage treatments induced by doxorubicin and TNF-alpha co-treatments

The present dataset contains the source data for Figure 2B of Tentner et al. (2012). The data shows the percentage of cultured cell-populations that stained positively and/or negatively for apoptotic markers cleaved caspase-3 and cleaved PARP, following DNA damage treatments induced by various doses of doxorubicin (0, 2 and 10 µmole/L) in the presence (100 ng/mL) or absence (0 ng/mL) of TNF-alpha co-treatment. For the six treatment conditions investigated, cell counts were made by flow cytometry at times 6, 12, 24, and 48 h following treatment; CULTURE DETAILS: U2OS cells were obtained from ATCC were maintained at 21% oxygen and 5% CO2 in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, 2mM L-glutamine, and used within 15-20 passages. The first thymidine block was released by washing the plates three times with PBS, and incubating them in fresh thymidine-free media for 12 h. A second thymidine block was then performed by re-addition of thymidine to 2.5 mM followed by incubation for an additional 18 h. Media was aspirated, plates were washed 3 with PBS, and replaced with fresh media in the presence or absence of 10 mM aphidicolin; ANALYSIS DETAILS: See supplementary journal publication; RESULT: The authors of the supplementary journal publication conclude that TNF enhances dose-dependent cell death following doxorubicin-induced DNA damage with minimal affect on dose-dependent cell-cycle arrest.

The crystal structure of human interferon β at 2.2-Å resolution

Type I interferons (IFNs) are helical cytokines that have diverse biological activities despite the fact that they appear to interact with the same receptor system. To achieve a better understanding of the structural basis for the different activities of α and β IFNs, we have determined the crystal structure of glycosylated human IFN-β at 2.2-Å resolution by molecular replacement. The molecule adopts a fold similar to that of the previously determined structures of murine IFN-β and human IFN-α2b but displays several distinct structural features. Like human IFN-α2b, human IFN-β contains a zinc-binding site at the interface of the two molecules in the asymmetric unit, raising the question of functional relevance for IFN-β dimers. However, unlike the human IFN-α2b dimer, in which homologous surfaces form the interface, human IFN-β dimerizes with contact surfaces from opposite sides of the molecule. The relevance of the structure to the effects of point mutations in IFN-β at specific exposed residues is discussed. A potential role of ligand–ligand interactions in the conformational assembly of IFN receptor components is discussed.

Microglia at brain stab wounds express connexin 43 and in vitro form functional gap junctions after treatment with interferon-γ and tumor necrosis factor-α

Gap junctional communication between microglia was investigated at rat brain stab wounds and in primary cultures of rat and mouse cells. Under resting conditions, rat microglia (FITC-isolectin-B4-reactive cells) were sparsely distributed in the neocortex, and most (95%) were not immunoreactive for Cx43, a gap junction protein subunit. At brain stab wounds, microglia progressively accumulated over several days and formed aggregates that frequently showed Cx43 immunoreactivity at interfaces between cells. In primary culture, microglia showed low levels of Cx43 determined by Western blotting, diffuse intracellular Cx43 immunoreactivity, and a low incidence of dye coupling. Treatment with the immunostimulant bacterial lipopolysaccharide (LPS) or the cytokines interferon-γ (INF-γ) or tumor necrosis factor-α (TNF-α) one at a time did not increase the incidence of dye coupling. However, microglia treated with INF-γ plus LPS showed a dramatic increase in dye coupling that was prevented by coapplication of an anti-TNF-α antibody, suggesting the release and autocrine action of TNF-α. Treatment with INF-γ plus TNF-α also greatly increased the incidence of dye coupling and the Cx43 levels with translocation of Cx43 to cell–cell contacts. The cytokine-induced dye coupling was reversibly inhibited by 18α-glycyrrhetinic acid, a gap junction blocker. Cultured mouse microglia also expressed Cx43 and developed dye coupling upon treatment with cytokines, but microglia from homozygous Cx43-deficient mice did not develop significant dye coupling after treatment with either INF-γ plus LPS or INF-γ plus TNF-α. This report demonstrates that microglia can communicate with each other through gap junctions that are induced by inflammatory cytokines, a process that may be important in the elaboration of the inflammatory response.

An essential role for the interferon-inducible, double-stranded RNA-activated protein kinase PKR in the tumor necrosis factor-induced apoptosis in U937 cells.

Tumor necrosis factor alpha (TNF-alpha) is well-characterized for its necrotic action against tumor cells; however, it has been increasingly associated with an apoptosis-inducing potential on target cells. While the signaling events and the actual cytolytic mechanism(s) for both TNF-alpha-induced necrosis and apoptosis remain to be fully elucidated, we report here on (i) the ability of TNF-alpha to induce apoptosis in the promonocytic U937 cells, (ii) the discovery of a cross-talk between the TNF-alpha and the interferon signaling pathways, and (iii) the pivotal role of interferon-inducible, double-stranded RNA-activated protein kinase (PKR) in the induction of apoptosis by TNF-alpha. Our data from microscopy studies, trypan blue exclusion staining, and apoptotic DNA ladder electrophoresis revealed that a subclone derived from U937 and carrying a PKR antisense expression vector was resistant to TNF-alpha-induced apoptosis. Further, TNF-alpha initiated a generalized RNA degradation process in which the participation of PKR was required. Finally, the PKR gene is a candidate "death gene" since overexpression of this gene could bring about apoptosis in U937 cells.

Cross-modulation by transforming growth factor beta in human tuberculosis: suppression of antigen-driven blastogenesis and interferon gamma production.

In tuberculosis, Mycobacterium tuberculosis (MTB)-stimulated T-cell responses are depressed transiently, whereas antibody levels are increased. Lymphoproliferative responses of peripheral blood mononuclear cells (PBMCs) from Pakistani tuberculosis (TB) patients to both mycobacterial and candidal antigens were suppressed by approximately 50% when compared to healthy purified protein derivative (PPD)-positive household contacts. Production of interferon gamma (IFN-gamma) in response to PPD also was depressed by 78%. Stimulation with PPD and the 30-kDa alpha antigen of MTB (30-kDa antigen) induced greater secretion of transforming growth factor beta (TGF-beta), but not interleukin 10 (IL-10) or tumor necrosis factor alpha (TNF-alpha), by PBMCs from TB patients compared to healthy contacts. The degree of suppression correlated with the duration of treatment; patients treated for <1 month had significantly lower T-cell blastogenesis and IFN-gamma production and higher levels of TGF-beta than did patients treated for >1 month. Neutralizing antibody to TGF-beta normalized lymphocyte proliferation in response to PPD, partially restored blastogenesis to candidal antigen, and significantly increased PPD-stimulated production of IFN-gamma in TB patients but not in contacts. Neutralizing antibody to IL-10 augmented, but did not normalize, T-cell responses to both PPD and candida in TB patients and candidal antigen in contacts. TGF-beta, produced in response to MTB antigens, therefore plays a prominent role in down-regulating potentially protective host effector mechanisms and looms as an important mediator of immunosuppression in TB.

Immunoregulatory properties of ISG15, an interferon-induced cytokine.

ISG15 is a 15-kDa protein of unique primary amino acid sequence, which is transcriptionally regulated by interferon (IFN) alpha and IFN-beta. Because it is synthesized in many cell types and secreted from human monocytes and lymphocytes, we postulated that ISG15 might act to modulate immune cell function. ISG15 stimulated B-depleted lymphocyte proliferation in a dose-dependent manner with significant proliferation induced by amounts of ISG15 as low as 1 ng/ml (58 pM). Maximal stimulation of [3H]thymidine incorporation by B-depleted lymphocytes occurred at 6-7 days. Immunophenotyping of ISG15-treated B-depleted lymphocyte cultures indicated a 26-fold expansion of natural killer (NK) cells (CD56+). In cytotoxicity assays, ISG15 was a potent inducer of cytolytic activity directed against both K562 (100 lytic units per 10(6) cells) and Daudi (80 lytic units per 10(6) cells) tumor cell targets, indicating that ISG15 enhanced lymphokine-activated killer-like activity. ISG15-induced NK cell proliferation required coculturing of T and NK cells, suggesting that soluble factor(s) were required. Measurement of ISG15-treated cell culture supernatants for cytokines indicated production of IFN-gamma (> 700 units/ml). No interleukin 2 or interleukin 12 was detected. IFN-gamma itself failed to stimulate lymphocyte proliferation and lymphokine-activated killer cell activation. Further, induced expression of IFN-gamma mRNA was detected by reverse transcription-PCR in T lymphocytes after ISG15 treatment but not in NK cells. Enhancement of NK cell proliferation, augmentation of non-major histocompatibility complex-restricted cytotoxicity, and induction of IFN-gamma from T cells identify ISG15 as a member of the cytokine cascade and suggest that it may be responsible for amplifying and directing some of the immunomodulatory effects of IFN-alpha or IFN-beta.

Ceramide formation during heat shock: a potential mediator of alpha B-crystallin transcription.

Ceramide has been identified as a potential second messenger that may mediate cell differentiation and apoptosis after exposure to hormonal agonists such as 1 alpha, 25-dihydroxyvitamin D3, tumor necrosis factor alpha, or gamma-interferon. The secondary cellular events that follow ceramide generation remain undefined. We report that in NIH WT-3T3 cells, ceramide induces an enhancement of gene transcription of alpha B-crystallin, a small heat shock protein. The levels of alpha B-crystallin, as measured by Northern blot and immunoblot analyses, were increased by the addition of an exogenous short-chain ceramide, N-acetylsphingosine, or by increasing endogenous intracellular ceramide by inhibition of glucosylceramide synthase. Similar effects were not seen in the expression of the closely related gene, Hsp25. To ascertain whether ceramide-mediated gene transcription was a feature of the heat shock response, cell ceramide was measured in heat shocked cells and observed to be elevated 2-fold immediately upon the return of cells to 37 degrees C. Thus ceramide formed after heat shock treatment of 3T3 cells may mediate the transcription events associated with the cell stress response.

Expression and signaling specificity of the IFNAR chain of the type I interferon receptor complex.

The IFNAR chain of the type I interferon (IFN) receptor (IFNIR) undergoes rapid ligand-dependent tyrosine phosphorylation and acts as a species-specific transducer for type I IFN action. Using the vaccinia/T7 expression system to amplify IFNAR expression, we found that human HeLa-S3 cells transiently express high levels of cell surface IFNAR chains (approximately 250,000 chains per cell). Metabolic labeling and immunoblot analysis of transfected HeLa cells show that the IFNAR chain is initially detected as 65-kDa and 98-kDa precursors, and then as the 130-kDa mature protein. Due to variation in N-glycosylation, the apparent molecular mass of the mature IFNAR chain varies from 105 to 135 kDa in different cells. IFNIR structure was characterized in various human cell lines by analyzing 125I-labeled IFN cross-linked complexes recognized by various antibodies against IFNIR subunits and JAK protein-tyrosine kinases. Precipitation of cross-linked material from Daudi cells with anti-IFNAR antibodies showed that IFNAR was present in a 240-kDa complex. Precipitation of cross-linked material from U937 cells with anti-TYK2 sera revealed a 240-kDa complex, which apparently did not contain IFNAR and was not present in IFN-resistant HEC1B cells. The tyrosine phosphorylation and down-regulation of the IFNAR chain were induced by type I IFN in several human cell lines of diverse origins but not in HEC1B cells. However, of type I IFNs, IFN-beta uniquely induced the tyrosine phosphorylation of a 105-kDa protein associated with the IFNAR chain in two lymphoblastoid cell lines (Daudi and U266), demonstrating the specificity of transmembrane signaling for IFN-beta and IFN-alpha through the IFNAR chain.

Crystal structure of the I-domain from the CD11a/CD18 (LFA-1, alpha L beta 2) integrin.

We report the 1.8-A crystal structure of the CD11a I-domain with bound manganese ion. The CD11a I-domain contains binding sites for intercellular adhesion molecules 1 and 3 and can exist in both low- and high-affinity states. The metal-bound form reported here is likely to represent a high-affinity state. The CD11a I-domain structure reveals a strained hydrophobic ridge adjacent to the bound metal ion that may serve as a ligand-binding surface and is likely to rearrange in the absence of bound metal ion. The CD11a I-domain is homologous to domains found in von Willebrand factor, and mapping of mutations found in types 2a and 2b von Willebrand disease onto this structure allows consideration of the molecular basis of these forms of the disease.

Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity.

The signaling mechanisms responsible for the induced expression of interferon (IFN) genes by viral infection or double-stranded RNA (dsRNA) are not well understood. Here we investigate the role of the interferon-induced dsRNA-dependent protein kinase PKR in the regulation of IFN induction. Biological activities attributed to PKR include regulating protein synthesis, mediating IFN actions, and functioning as a possible tumor suppressor. Since binding of dsRNA is required for its activation, PKR has been considered as a candidate signal transducer for regulating IFN expression. To examine this role of PKR, loss-of-function phenotypes in stable transformants of promonocytic U-937 cells were achieved by two different strategies, overexpression of an antisense PKR transcript or a dominant negative PKR mutant gene. Both types of PKR-deficient cells were more permissive for viral replication than the control U-937 cells. As the result of PKR loss, they also showed impaired induction of IFN-alpha and IFN-beta genes in response to several inducers--specifically, encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate. Interestingly, while IFN-alpha induction by dsRNA was impaired in PKR-deficient cells, IFN-beta induction remained intact. Loss of PKR function also resulted in decreased antiviral activity as elicited by IFN-alpha and, to a greater extent, by IFN-gamma. These results implicate PKR in the regulation of several antiviral activities.

Evidence of autocrine modulation of macrophage nitric oxide synthase by alpha-melanocyte-stimulating hormone.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a potent inhibitory agent in all major forms of inflammation. To identify a potential mechanism of antiinflammatory action of alpha-MSH, we tested its effects on production of nitric oxide (NO), believed to be a mediator common to all forms of inflammation. We measured NO and alpha-MSH production in RAW 264.7 cultured murine macrophages stimulated with bacterial lipopolysaccharide and interferon gamma. alpha-MSH inhibited production of NO, as estimated from nitrite production and nitration of endogenous macrophage proteins. This occurred through inhibition of production of NO synthase II protein; steady-state NO synthase II mRNA abundance was also reduced. alpha-MSH increased cAMP accumulation in RAW cells, characteristic of alpha-MSH receptors in other cell types. RAW cells also expressed mRNA for the primary alpha-MSH receptor (melanocortin 1). mRNA for proopiomelanocortin, the precursor molecular of alpha-MSH, was expressed in RAW cells, and tumor necrosis factor alpha increased production and release of alpha-MSH. These results suggest that the proinflammatory cytokine tumor necrosis factor alpha can induce macrophages to increase production of alpha-MSH, which then becomes available to act upon melanocortin receptors on the same cells. Such stimulation of melanocortin receptors could modulate inflammation by inhibiting the production of NO. The results suggest that alpha-MSH is an autocrine factor in macrophages which modulates inflammation by counteracting the effects of proinflammatory cytokines.