Although circulatory shock related to lethal toxin (LeTx) may play a primary role in lethality due to Bacillus anthracis infection, its mechanisms are unclear. We investigated whether LeTx-induced shock is associated with inflammatory cytokine and nitric oxide (NO) release. Sprague-Dawley rats with central venous and arterial catheters received 24-h infusions of LeTx (lethal factor 100 microg/kg; protective antigen 200 microg/kg) that produced death beginning at 9 h and a 7-day mortality rate of 53%. By 9 h, mean arterial blood pressure, heart rate, pH, and base excess were decreased and lactate and hemoglobin levels were increased in LeTx nonsurvivors compared with LeTx survivors and controls (diluent only) (P < or = 0.05 for each comparing the 3 groups). Despite these changes, arterial oxygen and circulating leukocytes and platelets were not decreased and TNF-alpha, IL-beta, IL-6, and IL-10 levels were not increased comparing either LeTx nonsurvivors or survivors to controls. Nitrate/nitrite levels and tissue histology also did not differ comparing LeTx animals and controls. In additional experiments, although 24-h infusions of LeTx and Escherichia coli LPS produced similar mortality rates (54 and 56%, respectively) and times to death (13.2 +/- 0.8 vs. 11.0 +/- 1.7 h, respectively) compared with controls, only LPS reduced circulating leukocytes, platelets, and IL-2 levels and increased TNF-alpha, IL-1 alpha and -1 beta, IL-6, IL-10, interferon-gamma, granulocyte macrophage-colony stimulating factor, RANTES, migratory inhibitory protein-1 alpha, -2, and -3, and monocyte chemotactic protein-1, as well as nitrate/nitrite levels (all P < or = 0.05 for the effects of LPS). Thus, in contrast to LPS, excessive inflammatory cytokine and NO release does not appear to contribute to the circulatory shock and lethality occurring with LeTx in this at model. Although therapies to modulate these host mediators may be applicable fo shock caused by LPS or other bacterial toxins, they may not with LeTx.

Dendritic cells (DCs) respond to microbial infections by undergoing phenotypic maturation and by producing multiple cytokines. In the present study, we analyzed the ability of influenza A and Sendai viruses to induce DC maturation and activate tumor necrosis factor alpha (TNF-alpha), alpha/beta interferon (IFN-alpha/beta), and IFN-like interleukin-28A/B (IFN-lambda2/3) and IL-29 (IFN-lambda1) gene expression in human monocyte-derived myeloid DCs (mDC). The ability of influenza A virus to induce mDC maturation or enhance the expression of TNF-alpha, IFN-alpha/beta, interleukin-28 (IL-28), and IL-29 genes was limited, whereas Sendai virus efficiently induced mDC maturation and enhanced cytokine gene expression. Influenza A virus-induced expression of TNF-alpha, IFN-alpha, IFN-beta, IL-28, and IL-29 genes was, however, dramatically enhanced when cells were pretreated with IFN-alpha. IFN-alpha priming led to increased expression of Toll-like receptor 3 (TLR3), TLR7, TLR8, MyD88, TRIF, and IFN regulatory factor 7 (IRF7) genes and enhanced influenza-induced phosphorylation and DNA binding of IRF3. Influenza A virus also enhanced the binding of NF-kappaB to the respective NF-kappaB elements of the promoters of IFN-beta and IL-29 genes. In mDC IL-29 induced MxA protein expression and possessed antiviral activity against influenza A virus, although this activity was lower than that of IFN-alpha or IFN-beta. Our results show that in human mDCs viruses can readily induce the expression of IL-28 and IL-29 genes whose gene products are likely to contribute to the host antiviral response.

Diabetes was induced in a normal nonautoimmune rat strain by rendering the animals relatively T cell deficient using a protocol of adult thymectomy and sublethal gamma irradiation. All male rats and 70% of females developed an acute syndrome with severe loss of weight and hyperglycemia. Diabetes in these lymphopoenic rats was associated with extensive insulitis involving CD4+ and CD8+ T cells and macrophages. The CD8+ T cells were essential for the development of diabetes but not insulitis. The autoimmune diabetes and insulitis were completely prevented by the injection of a particular CD4+ T cell subset, isolated from healthy syngeneic donors, of the phenotype CD45RClow T cell receptor alpha/beta+ RT6+ Thy-1- OX-40-. Cells of this protective phenotype, which make up about 5% of thoracic duct lymphocytes, were found to provide help for secondary antibody responses and produce interleukin 2 (IL-2) and IL-4, but no interferon gamma, on in vitro activation. These data provide evidence for the presence of autoreactive T cells in the normal immune system of the rat and reveal that in the intact animal these cells are prevented from expressing their autoreactive potential by other T cells.

2-5A-dependent RNase is the terminal factor in the interferon-regulated 2-5A system thought to function in both the molecular mechanism of interferon action and in the general control of RNA stability. However, direct evidence for specific functions of 2-5A-dependent RNase has been generally lacking. Therefore, we developed a strategy to block the 2-5A system using a truncated form of 2-5A-dependent RNase which retains 2-5A binding activity while lacking RNase activity. When the truncated RNase was stably expressed to high levels in murine cells, it prevented specific rRNA cleavage in response to 2-5A transfection and the cells were unresponsive to the antiviral activity of interferon alpha/beta for encephalomyocarditis virus. Remarkably, cells expressing the truncated RNase were also resistant to the antiproliferative activity of interferon. The truncated RNase is a dominant negative mutant that binds 2-5A and that may interfere with normal protein-protein interactions through nine ankyrin-like repeats.

Cytokine-specific monoclonal antibodies were used in enzyme-linked immunoadsorbant assays (ELISA) to examine a variety of synovial fluids for the presence of cytokines which might be expected to play some part in the pathology of arthritis. Low, but significant, levels of tumour necrosis factor alpha (TNF-alpha) were present in the majority of synovial fluids obtained from rheumatoid arthritis patients with a sero-positive history. Low levels of interferon (IFN)-alpha and IFN-gamma were also detected, but only IFN-alpha was significantly increased in the sero-positive group. Tumour necrosis factor beta (TNF-beta) was present only in trace amounts. These results suggest that the presence of cytokines, such as TNF-alpha and IFN-alpha in synovial fluid may be associated with tissue changes observed in rheumatoid joint disease and thus contribute to the pathology of the arthritis, but support evidence for the minimal role likely to be played by IFN-gamma in the joint pathology of rheumatoid arthritis.

The alpha/beta interferon (IFN-alpha/beta)-induced STAT signal transduction pathway leading to activation of the ISGF3 transcription complex and subsequent antiviral responses is the target of viral pathogenesis strategies. Members of the Rubulavirus genus of the Paramyxovirus family of RNA viruses have acquired the ability to specifically target either STAT1 or STAT2 for proteolytic degradation as a countermeasure for evading IFN responses. While type II human parainfluenza virus induces STAT2 degradation, simian virus 5 induces STAT1 degradation. The components of the IFN signaling system that are required for STAT protein degradation by these paramyxoviruses have been investigated in a series of human somatic cell lines deficient in IFN signaling proteins. Results indicate that neither the IFN-alpha/beta receptor, the tyrosine kinases Jak1 or Tyk2, nor the ISGF3 DNA-binding subunit, IFN regulatory factor 9 (IRF9), is required for STAT protein degradation induced by either virus. Nonetheless, both STAT1 and STAT2 are strictly required in the host cell to establish a degradation-permissive environment enabling both viruses to target their respective STAT protein. Complementation studies reveal that STAT protein-activating tyrosine phosphorylation and functional src homology 2 (SH2) domains are dispensable for creating a permissive STAT degradation environment in degradation-incompetent cells, but the N terminus of the missing STAT protein is essential. Protein-protein interaction analysis indicates that V and STAT proteins interact physically in vitro and in vivo. These results constitute genetic and biochemical evidence supporting a virus-induced, IFN-independent STAT protein degradation complex that contains at least STAT1 and STAT2.

The appearance of inflammatory markers associated with amyloid plaques indicates a state of chronic inflammation in Alzheimer's disease (AD). Multiple epidemiological studies also suggest that patients taking anti-inflammatory drugs have a decreased risk of developing AD. Here we present evidence that inflammatory cytokines can alter the metabolism of the beta-amyloid precursor protein (betaAPP). We show that the combination of tumor necrosis factor alpha and interferon gamma triggers the production of beta-amyloid peptides and inhibits the secretion of soluble APPs by human neuronal and extraneuronal cells. The results demonstrate a new mechanism by which inflammatory components can exacerbate the fundamental pathology in AD.

Toll-like receptors (TLRs) and retinoic acid-inducible gene I-like helicases (RLHs) are two major machineries recognizing RNA virus infection of innate immune cells. Intracellular signaling for TLRs and RLHs is mediated by their cytoplasmic adaptors, i.e., MyD88 or TRIF and IPS-1, respectively. In the present study, we investigated the contributions of TLRs and RLHs to the cytotoxic T-lymphocyte (CTL) response by using lymphocytoid choriomeningitis virus (LCMV) as a model virus. The generation of virus-specific cytotoxic T lymphocytes was critically dependent on MyD88 but not on IPS-1. Type I interferons (IFNs) are known to be important for the development of the CTL response to LCMV infection. Serum levels of type I IFNs and proinflammatory cytokines were mainly dependent on the presence of MyD88, although IPS-1(-/-) mice showed a decrease in IFN-alpha levels but not in IFN-beta and proinflammatory cytokine levels. Analysis of Ifna6(+/GFP) reporter mice revealed that plasmacytoid dendritic cells (DCs) are the major source of IFN-alpha in LCMV infection. MyD88(-/-) mice were highly susceptible to LCMV infection in vivo. These results suggest that recognition of LCMV by plasmacytoid DCs via TLRs is responsible for the production of type I IFNs in vivo. Furthermore, the activation of a MyD88-dependent innate mechanism induces a CTL response, which eventually leads to virus elimination.

Glutamate uptake by astrocytes has been postulated to play a neuroprotective role during brain inflammation. Using primary human fetal astrocyte cultures, we investigated the influence of selected cytokines on glutamate uptake activity. Interleukin (IL)-1beta and tumor necrosis factor-alpha dose-dependently inhibited astrocyte glutamate uptake, whereas interferon (IFN)-gamma alone stimulated this activity. The nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine, blocked IL-1beta-mediated inhibition of glutamate uptake, suggesting involvement of nitric oxide in the effect of IL-1beta. IL-1 receptor antagonist protein totally reversed the inhibitory effect of cytokines, suggesting a critical role of IL-1beta. The anti-inflammatory cytokine IFN-beta blocked cytokine (IL-1beta plus IFN-gamma)-induced inhibition of glutamate uptake with a corresponding reduction in nitric oxide generation. Taken together, these findings suggest that proinflammatory cytokines inhibit astrocyte glutamate uptake by a mechanism involving nitric oxide, and that IFN-beta may exert a therapeutically beneficial effect by blocking cytokine-induced nitric oxide production in inflammatory diseases of the brain.

Reverse cholesterol transport (RCT) is a pathway by which accumulated cholesterol is transported from the vessel wall to the liver for excretion, thus preventing atherosclerosis. Major constituents of RCT include acceptors such as high-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I), and enzymes such as lecithin:cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), hepatic lipase (HL) and cholesterol ester transfer protein (CETP). A critical part of RCT is cholesterol efflux, in which accumulated cholesterol is removed from macrophages in the subintima of the vessel wall by ATP-binding membrane cassette transporter A1 (ABCA1) or by other mechanisms, including passive diffusion, scavenger receptor B1 (SR-B1), caveolins and sterol 27-hydroxylase, and collected by HDL and apoA-I. Esterified cholesterol in the HDL is then delivered to the liver for excretion. In patients with mutated ABCA1 genes, RCT and cholesterol efflux are impaired and atherosclerosis is increased. In studies with transgenic mice, disruption of ABCA1 genes can induce atherosclerosis. Levels of HDL are inversely correlated with incidences of cardiovascular disease. Supplementation with HDL or apoA-I can reverse atherosclerosis by accelerating RCT and cholesterol efflux. On the other hand, pro-inflammatory factors such as interferon-gamma (IFN-gamma), endotoxin, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta), can be atherogenic by impairing RCT and cholesterol efflux, according to in vitro studies. RCT and cholesterol efflux play a major role in anti-atherogenesis, and modification of these processes may provide new therapeutic approaches to cardiovascular disease. Further research on new modifying factors for RCT and cholesterol efflux is warranted.

BACKGROUND

Genetic variations in cytokine genes are thought to regulate cytokine protein production. However, studies using T cell mitogens have not always demonstrated a significant relationship between cytokine polymorphisms and in vitro protein production. Furthermore, the functional consequence of a polymorphism at position -330 in the IL-2 gene has not been described. We associated in vitro protein production with cytokine gene polymorphic genotypes after costimulation of cultured peripheral blood lymphocytes.

METHODS

PBL were isolated from forty healthy volunteers. Cytokine protein production was assessed by enzyme-linked immunosorbent assay. Polymorphisms in interleukin- (IL) 2, IL-6, IL-10, tumor necrosis factor (TNF-alpha), tumor growth factor (TGF-beta), and interferon (IFN-gamma) were determined by polymerase chain reaction (PCR).

RESULTS

Statistical difference between protein production and cytokine polymorphic variants in the IL-10, IFN-gamma, and TNF-alpha genes was not evident after 48-hour stimulation with concanavalin-A. In contrast, after anti-CD3/CD28 stimulation significant differences (P<0.05) were found among high and low producers for IL-2, IL-6, and among high, intermediate, and low producers for IFN-gamma, and IL-10. Augmented levels of IL-2 in individuals that were homozygous for the polymorphic IL-2 allele were due to an early and sustained enhancement of IL-2 production. No association was found among TNF-alpha and TGF-beta genotypes and protein production.

CONCLUSIONS

Polymorphisms in IL-2, IL-6, IL-10, and IFN-gamma genes are associated with their protein production after anti-CD3/CD28 stimulation. The profound effect of the IL-2 gene polymorphism in homozygous individuals may serve as a marker for those that could mount the most vigorous allo- or autoimmune responses, or perhaps become tolerant more easily.

Type I diabetes is an autoimmune disease involving an interaction between an epigenetic event (possibly a viral infection), the pancreatic beta cells, and the immune system in a genetically susceptible host. The possibility that the type I interferons could mediate this interaction was tested with transgenic mice in which the insulin-producing beta cells expressed an interferon-alpha. These mice developed a hypoinsulinemic diabetes associated with a mixed inflammation centered on the islets. The inflammation and the diabetes were prevented with a neutralizing antibody to the interferon-alpha. Thus, the expression of interferon-alpha by the beta cells could be causal in the development of type I diabetes, which suggests a therapeutic approach to this disease.

Plasma lipid levels are elevated in people with diabetes, and a direct relationship can be demonstrated between indices of diabetic control and plasma lipid levels. Many observations suggest that diabetes may be associated with enhanced cytokine production, raising the possibility that some of the metabolic abnormalities associated with diabetes may be due to or exacerbated by cytokine overproduction. Tumor necrosis factor induces a rapid increase in serum triglyceride levels caused by an increase in VLDL of normal composition. Although in vitro studies showed that TNF decreases adipose tissue lipoprotein lipase activity, recent studies with intact animals demonstrated that TNF increases serum triglyceride levels by stimulating hepatic lipid secretion, not by affecting clearance. The increase in hepatic VLDL triglyceride secretion induced by TNF is due to both the stimulation of hepatic de novo fatty acid synthesis and an increase in lipolysis. Other cytokines including IL-1, IL-6, and alpha-interferon increase hepatic de novo fatty acid synthesis. Similarly, cytokines such as IL-1 and alpha-, beta-, and gamma-interferon also increase lipolysis. Thus, a variety of cytokines acting at different receptors can affect multiple processes that can alter lipid metabolism and increase serum lipid levels. These cytokine-induced increases in serum lipoprotein levels may be a beneficial response for the host. Studies show that lipoproteins, including VLDL, bind endotoxin and can protect against the toxic effects of endotoxin. Moreover, lipoproteins bind a variety of viruses, reducing their infectivity. Lipoproteins also bind urate crystals, which reduces the inflammatory response induced by these crystals.(ABSTRACT TRUNCATED AT 250 WORDS)

Structurally, the monocyte chemotactic proteins MCP-1, -2, and -3 form a subfamily of the C-C or beta-chemokines. Like other chemokines, MCPs are produced by a variety of cells on stimulation with cytokines (interleukin-1, tumor necrosis factor-alpha, interferon-gamma), bacterial and viral products or mitogens. MCP-1 levels are enhanced during infection and inflammation, which are characterized by leukocyte infiltration. In vitro, MCPs are chemotactic for a distinct spectrum of target cells and show different specific biological activities depending on the cell type and the chemokine tested. MCP-3 has the broadest range in that it activates monocytes, dendritic cells, lymphocytes, natural killer cells, eosinophils, basophils, and neutrophils. The most sensitive cells to all three MCPs are lymphocytes and monocytes. MCP-1 is a potent basophil activator but does not attract eosinophils, whereas, at higher concentrations, MCP-2 also stimulates both eosinophils and basophils. The signal transduction of MCPs on monocytes involves at least two G protein-linked C-C chemokine receptors: C-C CKR-1 binds MCP-3 and C-C CKR-2 binds MCP-1 and MCP-3 but not MCP-2. Receptor binding leads to enhanced [Ca2+]i for all chemokines except for MCP-2.

Since April 2012, there have been 17 laboratory-confirmed human cases of respiratory disease associated with newly recognized human betacoronavirus lineage C virus EMC (HCoV-EMC), and 7 of them were fatal. The transmissibility and pathogenesis of HCoV-EMC remain poorly understood, and elucidating its cellular tropism in human respiratory tissues will provide mechanistic insights into the key cellular targets for virus propagation and spread. We utilized ex vivo cultures of human bronchial and lung tissue specimens to investigate the tissue tropism and virus replication kinetics following experimental infection with HCoV-EMC compared with those following infection with human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus (SARS-CoV). The innate immune responses elicited by HCoV-EMC were also investigated. HCoV-EMC productively replicated in human bronchial and lung ex vivo organ cultures. While SARS-CoV productively replicated in lung tissue, replication in human bronchial tissue was limited. Immunohistochemistry revealed that HCoV-EMC infected nonciliated bronchial epithelium, bronchiolar epithelial cells, alveolar epithelial cells, and endothelial cells. Transmission electron microscopy showed virions within the cytoplasm of bronchial epithelial cells and budding virions from alveolar epithelial cells (type II). In contrast, there was minimal HCoV-229E infection in these tissues. HCoV-EMC failed to elicit strong type I or III interferon (IFN) or proinflammatory innate immune responses in ex vivo respiratory tissue cultures. Treatment of human lung tissue ex vivo organ cultures with type I IFNs (alpha and beta IFNs) at 1 h postinfection reduced the replication of HCoV-EMC, suggesting a potential therapeutic use of IFNs for treatment of human infection.

Since the first identification of interleukin (IL)-6 as a myeloma cell growth factor by Dr. Kawano's and Dr. Klein's groups 14 years ago, numerous studies have emphasized its major roles in the emergence of malignant plasma cells in vivo and in the generation of normal plasma cells. Four transcription factors control B-cell differentiation into plasma cells. The B-cell transcription factor pax-5 is mainly responsible for a B-cell phenotype, and bcl-6 represses the plasma cell transcription factor blimp-1 and plasma cell differentiation. bcl-6 expression is triggered by CD40 and IL-4 activation. A lack of CD40 and IL-4 activation yields a down-regulation of bcl-6 expression, and IL-6 stimulation yields an up-regulation of blimp-1, mainly through STAT3 activation. Blimp-1 further down-regulates bcl-6 and pax-5 expression and makes plasma cell differentiation possible. IL-6 as well as IL-10 up-regulate XBP-1. XBP-1 is another transcription factor that is involved in plasma cell differentiation and whose gene expression is shut down by pax-5. The plasma cell transcription factors blimp-1 and XBP-1 are up-regulated, and the B-cell transcription factors bcl-6 and pax-5 are down-regulated, in malignant cells compared to B-cells. Apart from the recent identification of these 4 transcription factors, the factors involved in normal plasma cell generation are mostly unknown. Regarding malignant plasma cells, 3 categories of growth factors have been identified: (1) the IL-6 family cytokines, IL-10, and interferon alpha that activate the Janus kinase-signal transducer and activator of transcription (JAK/STAT) and mitogen-activated protein (MAP) kinase pathways; (2) growth factors activating the phosphatidylinositol (PI)-3 kinase/AKT and MAP kinase pathways, unlike the JAK/STAT pathway (insulin-like growth factor 1, hepatocyte growth factor, and members of the epidermal growth factor family able to bind syndecan-1 proteoglycan); and (3) B-cell-activating factor (BAFF) or proliferation-inducing ligand (APRIL) that activate the nuclear factor KB and PI-3 kinase/AKT pathways. BAFF and APRIL bind to BAFF receptor and TACI and are major B-cell survival factors. Recent data indicate that these various growth factors may cooperate to provide optimum signaling because they are localized together and with cytoplasmic transduction elements in caveolinlinked membrane caveolae. The identification of these myeloma cell growth factors and of the associated transduction pathways should provide novel therapeutic targets in multiple myeloma.

Interferons are pleiotropic cytokines that exhibit negative regulatory effects on the growth of normal and malignant hematopoietic cells in vitro and in vivo. There are two different classes of interferons, Type I (alpha, beta, and omega) and Type II (gamma) interferons. Although the precise mechanisms by which these cytokines exhibit their potent effects on hematopoiesis remain unknown, there has been considerable progress in our understanding of the cellular changes that occur upon engagement of interferon receptors. It is now well established that Type I interferons activate multiple signaling pathways in hematopoietic cells, a finding consistent with their pleiotropic biological effects. One major pathway in Type I IFN signaling involves activation of Stat- proteins and formation of complexes that translocate to the nucleus and bind to specific elements to regulate gene transcription. The activation of this pathway (Jak-Stat pathway) is apparently regulated by members of the Jak-family of kinases, which are constitutively associated with the Type I IFN receptor. In addition to the Jak-Stat pathway, multiple other Jak-kinase-dependent signaling cascades are activated, including the IRS-PI 3'-kinase pathway, a pathway involving the vav proto-oncogene product, and a pathway involving adaptor proteins of the Crk-family (CrkL and CrkII). The only Type II interferon, IFNgamma, also activates multiple Jak-kinase-dependent signaling cascades, including the Stat and Crk pathways. Recent evidence suggests that non-Stat pathways play a critical role in the generation of signals for both Type I and Type II interferons and may be the primary mediators of their growth inhibitory effects on hematopoietic cells.

Granulation tissue fibroblasts (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle (SM) cells, including the presence of microfilament bundles and the expression of alpha-SM actin, the actin isoform present in SM cells and myoepithelial cells and particularly abundant in vascular SM cells. Myofibroblasts have been suggested to play a role in wound contraction and in retractile phenomena observed during fibrotic diseases. When contraction stops and the wound is fully epithelialized, myofibroblasts containing alpha-SM actin disappear, probably as a result of apoptosis, and the scar classically becomes less cellular and composed of typical fibroblasts with well-developed rough endoplasmic reticulum but with no more microfilaments. In contrast, alpha-SM actin expressing myofibroblasts persist in hypertrophic scars and in fibrotic lesions of many organs, including stroma reaction to epithelial tumours, where they are allegedly involved in retractile phenomena as well as in extracellular matrix accumulation. The mechanisms leading to the development of myofibroblastic features remain to be investigated. In vivo and in vitro investigations have shown that gamma-interferon exerts an antifibrotic activity at least in part by decreasing alpha-SM actin expression whereas heparin increases the proportion of alpha-SM actin positive cells. Recently, we have observed that the subcutaneous administration of transforming growth factor-beta 1 to rats results in the formation of a granulation tissue in which alpha-SM actin expressing myofibroblasts are particularly abundant. Other cytokines and growth factors, such as platelet-derived growth factor, basic fibroblast growth factor and tumour necrosis factor-alpha, despite their profibrotic activity, do not induce alpha-SM actin in myofibroblasts. In conclusion, fibroblastic cells are relatively undifferentiated and can assume a particular phenotype according to the physiological needs and/or the microenvironmental stimuli. Further studies on fibroblast adaptation phenomena appear to be useful for the understanding of the mechanisms of development and regression of pathological processes such as wound healing and fibrocontractive diseases.

Previous studies have demonstrated that Marburg viruses (MARV) and Ebola viruses (EBOV) inhibit interferon (IFN)-alpha/beta signaling but utilize different mechanisms. EBOV inhibits IFN signaling via its VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. In contrast, MARV infection inhibits IFNalpha/beta induced tyrosine phosphorylation of STAT1 and STAT2. MARV infection is now demonstrated to inhibit not only IFNalpha/beta but also IFNgamma-induced STAT phosphorylation and to inhibit the IFNalpha/beta and IFNgamma-induced tyrosine phosphorylation of upstream Janus (Jak) family kinases. Surprisingly, the MARV matrix protein VP40, not the MARV VP24 protein, has been identified to antagonize Jak and STAT tyrosine phosphorylation, to inhibit IFNalpha/beta or IFNgamma-induced gene expression and to inhibit the induction of an antiviral state by IFNalpha/beta. Global loss of STAT and Jak tyrosine phosphorylation in response to both IFNalpha/beta and IFNgamma is reminiscent of the phenotype seen in Jak1-null cells. Consistent with this model, MARV infection and MARV VP40 expression also inhibit the Jak1-dependent, IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. Finally, expression of MARV VP40 is able to prevent the tyrosine phosphorylation of Jak1, STAT1, STAT2 or STAT3 which occurs following over-expression of the Jak1 kinase. In contrast, MARV VP40 does not detectably inhibit the tyrosine phosphorylation of STAT2 or Tyk2 when Tyk2 is over-expressed. Mutation of the VP40 late domain, essential for efficient VP40 budding, has no detectable impact on inhibition of IFN signaling. This study shows that MARV inhibits IFN signaling by a mechanism different from that employed by the related EBOV. It identifies a novel function for the MARV VP40 protein and suggests that MARV may globally inhibit Jak1-dependent cytokine signaling.

The present study investigates mechanisms involved in cooperation between IFN-gamma and TNF-alpha to promote transcription from the IP-10 gene in NIH 3T3 cells. IFN-gamma synergistically enhanced TNF-alpha-induced levels of IP-10 mRNA, whereas levels of JE (MCP-1) or KC (GRO/MGSA) mRNA induced by TNF-alpha were unaffected by IFN-gamma. The cooperation between IFN-gamma and TNF-alpha for induction of IP-10 mRNA was independent of de novo protein synthesis and mediated at least in part by increased transcription. Transient transfection analysis with a 243-bp fragment flanking the transcription start site of the murine IP-10 gene indicated that synergy between the two stimuli was dependent upon occupancy of at least two of three critical regulatory sequence elements: an IFN-stimulated response element (ISRE) and one of two kappa B sites. IFN-gamma and TNF-alpha independently activated nuclear factors capable of specific interaction with the ISRE and kappa B sites, respectively. IFN-gamma induced two ISRE binding complexes, one of which was protein synthesis independent, appeared within 15 min of stimulation, and contained p91 or signal transducer and activator of transcription (STAT) 1. TNF-alpha induced only one ISRE binding activity, which was dependent upon protein synthesis. TNF-alpha also induced kappa B binding activity that was composed of NF-kappa BB binding activity. Together these results indicate that the highly synergistic transcriptional activation of the IP-10 gene by IFN-gamma and TNF-alpha involves the cooperation between factors that are independently activated by the two stimuli and that bind to independent sites.

Apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) is a potent inhibitor of infection by a wide range of retroviruses. Although recent reports have suggested that human APOBEC3G exerts antiviral activity against hepatitis B virus, APOBEC3G expression is normally low in the human liver. To clarify the role of APOBEC3G in cellular defenses against hepatitis viruses, the regulation of the APOBEC3G expression was investigated in human hepatocytes. Endogenous transcripts of nine APOBEC family members were barely detectable in quiescent liver cells. However, APOBEC3G was significantly up-regulated in response to interferon-alpha (IFN-alpha) stimulation in HepG2, Huh-7, and primary human hepatocytes. IFN regulatory factor elements that are important for IFN-inducible promoter activity were identified 5' upstream from the human APOBEC3G gene. Our findings provided the first evidence showing that APOBEC3G is induced by IFN stimulation in human hepatocytes and thus could be involved in host defense mechanisms directed against hepatitis viruses.

Interferon establishes an antiviral state in numerous cell types through the induction of a set of immediate-early response genes. Activation of these genes is mediated by phosphorylation of latent transcription factors of the STAT family. We found that infection of primary foreskin fibroblasts with human cytomegalovirus (HCMV) causes selective transcriptional activation of the alpha/beta-interferon-responsive ISG54 gene. However, no activation or nuclear translocation of STAT proteins was detected. Activation of ISG54 occurs independent of protein synthesis but is prevented by protein tyrosine kinase inhibitors. Further analysis revealed that HCMV infection induced the DNA binding of a novel complex, tentatively called cytomegalovirus-induced interferon-stimulated response element binding factor (CIF). CIF is composed, at least in part, of the recently identified interferon regulatory factor 3 (IRF3), but it does not contain the STAT1 and STAT2 proteins that participate in the formation of interferon-stimulated gene factor 3. IRF3, which has previously been shown to possess no intrinsic transcriptional activation potential, interacts with the transcriptional coactivator CREB binding protein, but not with p300, to form CIF. Activating interferon-stimulated genes without the need for prior synthesis of interferons might provide the host cell with a potential shortcut in the activation of its antiviral defense.

Murine hybridomas producing monoclonal antibodies (mAb) specific to human interleukin 6 (IL 6/BSF-2) were established. One of these hybridomas (MH60.BSF2) was found to be dependent on IL 6 for its in vitro growth. None of the known biological factors tested, such as recombinant (r) human (Hu) IL 1 alpha, rHuIL 1 beta, rHuIL 2, rHuIL 3, rHuIL 4, rHu interferon (IFN)-gamma, HuIFN-beta, rHuG-CSF, or recombinant murine (Mu) IL 3, MuIL 4, rMuIL 5, could induce the in vitro growth of MH60.BSF2 cells. The half-maximum tritiated thymidine uptake by MH60.BSF2 cells could be achieved by picogram amounts of rIL 6, making this hybridoma clone an indicator cell for specific and sensitive detection of the IL 6 activity in test samples. The MH166.BSF2 clone was found to produce IgG1,chi type mAb (alpha BSF2-166) capable of neutralizing IL 6 activity. The other clone, MH60.BSF2, produced IgM,chi type mAb (alpha BSF2-60) unable to neutralize IL 6 activity. Both mAb specifically reacted with IL 6 as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis. An enzyme-linked immunosorbent assay (ELISA) utilizing alpha BSF2-166 and rabbit anti-IL 6 antibodies was established which could detect as low as 50 pg/ml of IL 6. Since both the ELISA and MH60.BSF2 hybridoma could detect small amounts of IL 6 in biological fluids, they constitute powerful tools in exploring the presence or the role of IL 6 in various immunological disorders.

The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), cause frequently lethal viral hemorrhagic fever. These infections induce potent cytokine production, yet these host responses fail to prevent systemic virus replication. Consistent with this, filoviruses have been found to encode proteins VP35 and VP24 that block host interferon (IFN)-alpha/beta production and inhibit signaling downstream of the IFN-alpha/beta and the IFN-gamma receptors, respectively. VP35, which is a component of the viral nucleocapsid complex and plays an essential role in viral RNA synthesis, acts as a pseudosubstrate for the cellular kinases IKK-epsilon and TBK-1, which phosphorylate and activate interferon regulatory factor 3 (IRF-3) and interferon regulatory factor 7 (IRF-7). VP35 also promotes SUMOylation of IRF-7, repressing IFN gene transcription. In addition, VP35 is a dsRNA-binding protein, and mutations that disrupt dsRNA binding impair VP35 IFN-antagonist activity while leaving its RNA replication functions intact. The phenotypes of recombinant EBOV bearing mutant VP35s unable to inhibit IFN-alpha/beta demonstrate that VP35 IFN-antagonist activity is critical for full virulence of these lethal pathogens. The structure of the VP35 dsRNA-binding domain, which has recently become available, is expected to provide insight into how VP35 IFN-antagonist and dsRNA-binding functions are related. The EBOV VP24 protein inhibits IFN signaling through an interaction with select host cell karyopherin-alpha proteins, preventing the nuclear import of otherwise activated STAT1. It remains to be determined to what extent VP24 may also modulate the nuclear import of other host cell factors and to what extent this may influence the outcome of infection. Notably, the Marburg virus VP24 protein does not detectably block STAT1 nuclear import, and, unlike EBOV, MARV infection inhibits STAT1 and STAT2 phosphorylation. Thus, despite their similarities, there are fundamental differences by which these deadly viruses counteract the IFN system. It will be of interest to determine how these differences influence pathogenesis.