T helper cell type 1 (Th1) and 2 (Th2) are central to immune regulation. However, no stable cell surface marker capable of distinguishing and separating these two subsets of CD4(+) cells has yet been found. Using differential display PCR, we have identified a gene encoding a cell membrane bound molecule, originally designated ST2L, T1, DER4, or Fit, expressed constitutively and stably on the surface of murine Th2s, but not Th1s even after stimulation with a range of immunological stimuli. Antibody against a peptide derived from ST2L strongly and stably labeled the surface of cloned Th2s but not Th1s, and Th2s but not Th1s derived from naive T cells of ovalbumin T cell receptor-alpha/beta transgenic mice. Three-color single cell flow cytometric analysis shows that cell surface ST2L coexpressed with intracellular interleukin (IL)-4, but not with interferon (IFN)-gamma. The antibody selectively lysed Th2s in vitro in a complement-dependent manner. In vivo, it enhanced Th1 responses by increasing IFN-gamma production and decreasing IL-4 and IL-5 synthesis. It induced resistance to Leishmania major infection in BALB/c mice and exacerbated collagen-induced arthritis in DBA/1 mice. Thus, ST2L is a stable marker distinguishing Th2s from Th1s and is also associated with Th2 functions. Hence, it may be a target for therapeutic intervention.

To study the capacity of human neurons to mount innate immunity responses to viral infections, we infected cells of a human postmitotic neuron-derivative cell line, NT2-N, with rabies virus (RABV) and herpes simplex type 1 (HSV-1). Changes in neuronal gene expression were analyzed by use of Affymetrix microarrays. Applying a twofold cutoff, RABV increased the transcription of 228 genes, and HSV-1 increased the transcription of 263 genes. The most striking difference between the two infections concerns genes involved in immunity. These genes represent 24% of the RABV-upregulated genes and only 4.9% of the HSV-1-upregulated genes. Following RABV infection, the most upregulated genes belong to the immunity cluster and included almost exclusively genes for beta interferon (IFN-beta) primary and secondary responses as well as genes for chemokines (CCL-5, CXCL-10) and inflammatory cytokines (interleukin 6 [IL-6], tumor necrosis factor alpha, interleukin 1 alpha). In contrast, HSV-1 infection did not increase IFN-beta gene transcripts and triggered the production of only IL-6 and interferon regulatory factor 1 mRNAs. The microarray results were confirmed by real-time PCR, immunocytochemistry, and enzyme-linked immunosorbent assay. Human neurons were found to express Toll-like receptor 3. They produced IFN-beta after treatment with poly(I:C) but not with lipopolysaccharide. Thus, human neurons can mount an innate immunity response to double-stranded RNA. These observations firmly establish that human neurons, in absence of glia, have the intrinsic machinery to sense virus infection.

The intercellular adhesion molecule (ICAM) 1 is an Ig-like cell adhesion molecule expressed by several cell types, including leukocytes and endothelial cells. It can be induced in a cell-specific manner by several cytokines, for example, tumor necrosis factor-alpha, interleukin-1, and interferon-gamma, and inhibited by glucocorticoids. Its ligands are the membrane-bound integrin receptors LFA-1 and Mac-1 on leukocytes, CD43, the soluble molecule fibrinogen, the matrix factor hyaluronan, rhinoviruses, and Plasmodium falciparum malaria-infected erythrocytes. ICAM-1 expression is predominantly transcriptionally regulated. The ICAM-1 promoter contains several enhancer elements, among them a novel kappa B element which mediates effects of 12-O-tetradecanoylphorbol-13-acetate, interleukin-1, lipopolysaccharide, tumor necrosis factor-alpha, and glucocorticoids. Expression regulation is cell specific and depends on the availability of cytokine/hormone receptors, signal transduction pathways, transcription factors, and posttranscriptional modification. ICAM-1 plays a role in inflammatory processes and in the T-cell mediated host defense system. It functions as a costimulatory molecule on antigen-presenting cells to activate MHC class II restricted T-cells, and on other cell types in association with MHC class I to activate cytotoxic T-cells. ICAM-1 on endothelium plays an important role in migration of (activated) leukocytes to sites of inflammation. ICAM-1 is shed by the cell and detected in plasma as sICAM-1. Regulation and significance of sICAM-1 are as yet unclear, but sICAM-1 is increased in many pathological conditions. ICAM-1 may play a pathogenetic role in rhinovirus infections. Derangement of ICAM-1 expression probably contributes to the clinical manifestations of a variety of diseases, predominantly by interfering with normal immune function. Among these are malignancies (e.g., melanoma and lymphomas), many inflammatory disorders (e.g., asthma and autoimmune disorders), atherosclerosis, ischemia, certain neurological disorders, and allogeneic organ transplantation. Interference with ICAM-1 leukocyte interaction using mAbs, soluble ICAM-1, antisense ICAM-1 RNA, and in the case of melanoma mAb-coupled immunotoxin, may offer therapeutic possibilities in the future. Integration of knowledge concerning membrane-bound and soluble ICAM-1 into a single functional system is likely to contribute to elucidating the immunoregulatory function of ICAM-1 and its pathophysiological significance in various disease entities.

The discovery and initial description of the interferon-lambda (IFN-lambda) family in early 2003 opened an exciting new chapter in the field of IFN research. There are 3 IFN-lambda genes that encode 3 distinct but highly related proteins denoted IFN-lambda1, -lambda2, and -lambda3. These proteins are also known as interleukin-29 (IL-29), IL-28A, and IL-28B, respectively. Collectively, these 3 cytokines comprise the type III subset of IFNs. They are distinct from both type I and type II IFNs for a number of reasons, including the fact that they signal through a heterodimeric receptor complex that is different from the receptors used by type I or type II IFNs. Although type I IFNs (IFN-alpha/beta) and type III IFNs (IFN-lambda) signal via distinct receptor complexes, they activate the same intracellular signaling pathway and many of the same biological activities, including antiviral activity, in a wide variety of target cells. Consistent with their antiviral activity, expression of the IFN-lambda genes and their corresponding proteins is inducible by infection with many types of viruses. Therefore, expression of the type III IFNs (IFN-lambdas) and their primary biological activity are very similar to the type I IFNs. However, unlike IFN-alpha receptors which are broadly expressed on most cell types, including leukocytes, IFN-lambda receptors are largely restricted to cells of epithelial origin. The potential clinical importance of IFN-lambda as a novel antiviral therapeutic agent is already apparent. In addition, preclinical studies by several groups indicate that IFN-lambda may also be useful as a potential therapeutic agent for other clinical indications, including certain types of cancer.

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G), a cytidine deaminase, is a recently recognized innate intracellular protein with lethal activity against human immunodeficiency virus (HIV). Packaged into progeny virions, APOBEC3G enzymatic activity leads to HIV DNA degradation. As a counterattack, HIV virion infectivity factor (Vif) targets APOBEC3G for proteasomal proteolysis to exclude it from budding virions. Based on the ability of APOBEC3G to antagonize HIV infection, considerable interest hinges on elucidating its mechanism(s) of regulation. In this study, we provide the first evidence that an innate, endogenous host defense factor has the potential to promote APOBEC3G and rebuke the virus-mediated attempt to control its cellular host. We identify interferon (IFN)-alpha as a potent inducer of APOBEC3G to override HIV Vif neutralization of APOBEC3 proteins that pose a threat to efficient macrophage HIV replication. Our data provide a new dimension by which IFN-alpha mediates its antiviral activity and suggest a means to render the host nonpermissive for viral replication.

The liver lobule is formed by parenchymal cells, i.e., hepatocytes and nonparenchymal cells. In contrast to hepatocytes that occupy almost 80% of the total liver volume and perform the majority of numerous liver functions, nonparenchymal liver cells, which contribute only 6.5% to the liver volume, but 40% to the total number of liver cells, are localized in the sinusoidal compartment of the tissue. The walls of hepatic sinusoid are lined by three different cell types: sinusoidal endothelial cells (SEC), Kupffer cells (KC), and hepatic stellate cells (HSC, formerly known as fat-storing cells, Ito cells, lipocytes, perisinusoidal cells, or vitamin A-rich cells). Additionally, intrahepatic lymphocytes (IHL), including pit cells, i.e., liver-specific natural killer cells, are often present in the sinusoidal lumen. It has been increasingly recognized that both under normal and pathological conditions, many hepatocyte functions are regulated by substances released from neighboring nonparenchymal cells. Liver sinusoidal endothelial cells constitute the lining or wall of the hepatic sinusoid. They perform important filtration function due to the presence of small fenestrations that allow free diffusion of many substances, but not of particles of the size of chylomicrons, between the blood and the hepatocyte surface. SEC show huge endocytic capacity for many ligands including glycoproteins, components of the extracellular matrix (ECM; such as hyaluronate, collagen fragments, fibronectin, or chondroitin sulphate proteoglycan), immune complexes, transferrin and ceruloplasmin. SEC may function as antigen-presenting cells (APC) in the context of both MHC-I and MHC-II restriction with the resulting development of antigen-specific T-cell tolerance. They are also active in the secretion of cytokines, eicosanoids (i.e., prostanoids and leukotrienes), endothelin-1, nitric oxide, and some ECM components. Kupffer cells are intrasinusoidally located tissue macrophages with a pronounced endocytic and phagocytic capacity. They are in constant contact with gut-derived particulate materials and soluble bacterial products so that a subthreshold level of their activation in the normal liver may be anticipated. Hepatic macrophages secrete potent mediators of the inflammatory response (reactive oxygen species, eicosanoids, nitric oxide, carbon monoxide, TNF-alpha, and other cytokines), and thus control the early phase of liver inflammation, playing an important part in innate immune defense. High exposure of Kupffer cells to bacterial products, especially endotoxin (lipopolysaccharide, LPS), can lead to the intensive production of inflammatory mediators, and ultimately to liver injury. Besides typical macrophage activities, Kupffer cells play an important role in the clearance of senescent and damaged erythrocytes. Liver macrophages modulate immune responses via antigen presentation, suppression of T-cell activation by antigen-presenting sinusoidal endothelial cells via paracrine actions of IL-10, prostanoids, and TNF-alpha, and participation in the development of oral tolerance to bacterial superantigens. Moreover, during liver injury and inflammation, Kupffer cells secrete enzymes and cytokines that may damage hepatocytes, and are active in the remodeling of extracellular matrix. Hepatic stellate cells are present in the perisinusoidal space. They are characterized by abundance of intracytoplasmic fat droplets and the presence of well-branched cytoplasmic processes, which embrace endothelial cells and provide focally a double lining for sinusoid. In the normal liver HSC store vitamin A, control turnover of extracellular matrix, and regulate the contractility of sinusoids. Acute damage to hepatocytes activates transformation of quiescent stellate cells into myofibroblast-like cells that play a key role in the development of inflammatory fibrotic response. Pit cells represent a liver-associated population of large granular lymphocytes, i.e., natural killer (NK) cells. They spontaneously kill a variety of tumor cells in an MHC-unrestricted way, and this antitumor activity may be enhanced by the secretion of interferon-gamma. Besides pit cells, the adult liver contains other subpopulations of lymphocytes such as gamma delta T cells, and both "conventional" and "unconventional" alpha beta T cells, the latter containing liver-specific NK T cells. The development of methods for the isolation and culture of main liver cell types allowed to demonstrate that both nonparenchymal and parenchymal cells secrete tens of mediators that exert multiple paracrine and autocrine actions. Co-culture experiments and analyses of the effects of conditioned media on cultures of another liver cell type have enabled the identification of many substances released from non-parenchymal liver cells that evidently regulate some important functions of neighboring hepatocytes and non-hepatocytes. To the key mediators involved in the intercellular communication in the liver belong prostanoids, nitric oxide, endothelin-1, TNF-alpha, interleukins, and chemokines, many growth factors (TGF-beta, PDGF, IGF-I, HGF), and reactive oxygen species (ROS). Paradoxically, the cooperation of liver cells is better understood under some pathological conditions (i.e., in experimental models of liver injury) than in normal liver due to the possibility of comparing cellular phenotype under in vivo and in vitro conditions with the functions of the injured organ. The regulation of vitamin A metabolism provides an example of the physiological role for cellular cross-talk in the normal liver. The majority (up to 80%) of the total body vitamin A is stored in the liver as long-chain fatty acid esters of retinal, serving as the main source of retinoids that are utilized by all tissues throughout the body. Hepatocytes are directly involved in the uptake from blood of chylomicron remnants, and the synthesis of retinol-binding protein that transfers retinol to other tissues. However, more than 80% of the liver retinoids are stored in lipid droplets of hepatic stellate cells. HSC are capable of both uptake and release of retinol depending on the body's retinol status. The activity of some major enzymes of vitamin A metabolism have been found to be many times higher per protein basis in stellate cells than in hepatocytes. Despite progress in the understanding of the roles played by these two cell types in hepatic retinoid metabolism, the way in which retinoids move between the parenchymal cells, stellate cells, and blood plasma has not been fully elucidated. Sinusoidal blood flow is, to a great extent, regulated by hepatic stellate cells that can contract due to the presence of smooth muscle alpha-actin. The main vasoactive substances that affect constriction or relaxation of HSC derive both from distant sources and from neighboring hepatocytes (carbon monoxide, leukotrienes), endothelial cells (endothelin, nitric oxide, prostaglandins), Kupffer cells (prostaglandins, NO), and stellate cells themselves (endothelin, NO). The cellular cross-talk reflected by the fine-tuned modulation of sinusoidal contraction becomes disturbed under pathological conditions, such as endotoxemia or liver fibrosis, through the excess synthesis of vasoregulatory compounds and the involvement of additional mediators acting in a paracrine way. The liver is an important source of some growth factors and growth factor-binding proteins. Although hepatocytes synthesize the bulk of insulin-like growth factor I (IGF-I), also other types of nonparenchymal liver cells may produce this peptide. Cell-specific expression of distinct IGF-binding proteins observed in the rat and human liver provides the potential for specific regulation of hepatic IGF-I synthesis not only by growth hormone, insulin, and IGF-I, but also by cytokines released from activated Kupffer (IL-1, TNF-alpha, TGF-beta) or stellate cells (TGF-alpha, TGF-beta). Hepatic stellate cells may affect turnover of hepatocytes through the synthesis of potent positive as well as negative signals such as, respectively, hepatocyte-growth-factor or TGF-beta. Although hepatocytes seem not to produce TGF-beta, a pleiotropic cytokine synthesized and secreted in the latent form by Kupffer and stellate cells, they may contribute to its actions in the liver by the intracellular activation of latent TGF-beta, and secretion of the biologically active isoform. Many mediators that reach the liver during inflammatory processes, such as endotoxins, immune-complexes, anaphylatoxins, and PAF, increase glucose output in the perfused liver, but fail to do so in isolated hepatocytes, acting indirectly via prostaglandins released from Kupffer cells. In the liver, prostaglandins synthesized from arachidonic acid mainly in Kupffer cells in a response to various inflammatory stimuli, modulate hepatic glucose metabolism by increasing glycogenolysis in adjacent hepatocytes. The release of glucose from glycogen supports the increased demand for energetic fuel by the inflammatory cells such as leukocytes, and additionally enables enhanced glucose turnover in sinusoidal endothelial cells and Kupffer cells which is necessary for effective defense of these cells against invading microorganisms and oxidative stress in the liver. Leukotrienes, another oxidation product of arachidonic acid, have vasoconstrictive, cholestatic, and metabolic effects in the liver. A transcellular synthesis of cysteinyl leukotrienes (LTC4, LTD4, and LTE4) functions in the liver: LTA4, an important intermediate, is synthesized in Kupffer cells, taken up by hepatocytes, converted into the potent LTC4, and then released into extracellular space, acting in a paracrine way on Kupffer and sinusoidal endothelial cells. Thus, hepatocytes are target cells for the action of eicosanoids and the site of their transformation and degradation, but can not directly oxidate arachidonic acid to eicosanoids. (ABSTRACT TRUNCATED)

The immunologic privilege of the central nervous system (CNS) makes it crucial that CNS resident cells be capable of responding rapidly to infection. Astrocytes have been reported to express Toll-like receptors (TLRs), hallmark pattern recognition receptors of the innate immune system, and respond to their ligation with cytokine production. Astrocytes have also been reported to respond to cytokines of the adaptive immune system with the induction of antigen presentation functions. Here we have compared the ability of TLR stimuli and the adaptive immune cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) to induce a variety of immunologic functions of astrocytes. We show that innate signals LPS- and poly I:C lead to stronger upregulation of TLRs and production of the cytokines IL-6 and TNF-alpha as well as innate immune effector molecules IFN-alphabeta, and iNOS compared with cytokine-stimulated astrocytes. Both innate stimulation and adaptive stimulation induce similar expression of the chemokines CCL2, CCL3, and CCL5, as well as similar enhancement of adhesion molecule ICAM-1 and VCAM-1 expression by astrocytes. Stimulation with adaptive immune cytokines, however, was unique in its ability to induce upregulation of MHC II and the functional ability of astrocytes to activate CD4(+) T cells. These results indicate potentially important and changing roles for astrocytes during the progression of CNS infection.

Type beta transforming growth factor (TGF-beta) is a unique polypeptide that has been isolated from a number of different tissues and can induce the phenotypic transformation of non-neoplastic fibroblasts as measured by the stimulation of their growth in soft agar. Recently, TGF-beta has been demonstrated to exert profound inhibitory effects on T and B lymphocyte proliferation. In this study, the effects of TGF-beta on natural killer (NK) cell function were investigated. After 20 hr of culture in the presence of TGF-beta, the NK activity of peripheral blood lymphocytes (PBL) was significantly reduced compared with PBL cultured in medium alone. Similarly, TGF-beta produced a significant depression in the cytolytic activity of highly enriched large granular lymphocytes (LGL). This effect of TGF-beta appeared to be mediated directly on the effector cells, because cultivation of the K562 target cells in TGF-beta did not affect target cell susceptibility to lysis. Binding studies with 125I-TGF-beta indicated that LGL possess approximately 1400 high-affinity (Kd = 1PM) receptors/cell, which represents a considerably higher affinity receptor for TGF-beta than that found on fibroblasts. Culturing of PBL and LGL in TGF-beta resulted in a marked blunting of the boosting of NK cytolysis by interferon-alpha but not by interleukin 2, which suggested that TGF-beta may down-regulate interferon-alpha receptors on NK cells. These results, indicate that in addition to inhibitory effects on T and B cells, TGF-beta also inhibits NK cell function. Although the in vivo role of TGF-beta is presently undefined, it may be an important immunoregulatory protein that has a negative influence on lymphocyte activation.

Interleukin-6 (IL-6, also known as B-cell stimulatory factor 2/interferon beta 2) was previously shown to support the proliferation of granulocyte/macrophage progenitors and indirectly support the formation of multilineage and blast cell colonies in cultures of spleen cells from normal mice. We report here that IL-3 and IL-6 act synergistically in support of the proliferation of murine multipotential progenitors in culture. The time course of total colony formation by spleen cells isolated from mice 4 days after injection of 5-fluorouracil (150 mg/kg) was significantly shortened in cultures containing both lymphokines relative to cultures supported by either of the two factors. Serial observations (mapping) of individual blast cell colonies in culture revealed that blast cell colonies emerged after random time intervals in the presence of IL-3. The average time of appearance in IL-6 alone was somewhat delayed, and in cultures containing both factors the appearance of multilineage blast cell colonies was significantly hastened relative to cultures grown in the presence of the individual lymphokines. In cultures of day-2 post-5-fluorouracil bone marrow cells, IL-6 failed to support colony formation; IL-3 alone supported the formation of a few granulocyte/macrophage colonies, but the combination of factors acted synergistically to yield multilineage and a variety of other types of colonies. In this system, IL-1 alpha also acted synergistically with IL-3, but the effect was smaller, and no multilineage colonies were seen. Together these results indicate that IL-3 and IL-6 act synergistically to support the proliferation of hemopoietic progenitors and that at least part of the effect results from a decrease in the G0 period of the individual stem cells.

The current armamentarium for the chemotherapy of viral infections consists of 37 licensed antiviral drugs. For the treatment of human immunodeficiency virus (HIV) infections, 19 compounds have been formally approved: (i) the nucleoside reverse transcriptase inhibitors (NRTIs) zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir and emtricitabine; (ii) the nucleotide reverse transcriptase inhibitor (NtRTI) tenofovir disoproxil fumarate; (iii) the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine, delavirdine and efavirenz; (iv) the protease inhibitors saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir (combined with ritonavir at a 4/1 ratio) and atazanavir; and the viral entry inhibitor enfuvirtide. For the treatment of chronic hepatitis B virus (HBV) infections, lamivudine as well as adefovir dipivoxil have been approved. Among the anti-herpesvirus agents, acyclovir, valaciclovir, penciclovir (when applied topically), famciclovir, idoxuridine and trifluridine (both applied topically) as well as brivudin are used in the treatment of herpes simplex virus (HSV) and/or varicella-zoster virus (VZV) infections; and ganciclovir, valganciclovir, foscarnet, cidofovir and fomivirsen (the latter upon intravitreal injection) have proven useful in the treatment of cytomegalovirus (CMV) infections in immunosuppressed patients (i.e. AIDS patients with CMV retinitis). Following amantadine and rimantadine, the neuraminidase inhibitors zanamivir and oseltamivir have recently become available for the therapy (and prophylaxis) of influenza virus infections. Ribavirin has been used (topically, as aerosol) in the treatment of respiratory syncytial virus (RSV) infections, and the combination of ribavirin with (pegylated) interferon-alpha has received increased acceptance for the treatment of hepatitis C virus (HCV) infections.

Virus-infected cells secrete a broad range of interferons (IFN) which confer resistance to yet uninfected cells by triggering the synthesis of antiviral factors. The relative contributions of the various IFN subtypes to innate immunity against virus infections remain elusive. IFN-alpha, IFN-beta, and other type I IFN molecules signal through a common, universally expressed cell surface receptor, whereas type III IFN (IFN-lambda) uses a distinct cell-type-specific receptor complex for signaling. Using mice lacking functional receptors for type I IFN, type III IFN, or both, we found that IFN-lambda plays an important role in the defense against several human pathogens that infect the respiratory tract, such as influenza A virus, influenza B virus, respiratory syncytial virus, human metapneumovirus, and severe acute respiratory syndrome (SARS) coronavirus. These viruses were more pathogenic and replicated to higher titers in the lungs of mice lacking both IFN receptors than in mice with single IFN receptor defects. In contrast, Lassa fever virus, which infects via the respiratory tract but primarily replicates in the liver, was not influenced by the IFN-lambda receptor defect. Careful analysis revealed that expression of functional IFN-lambda receptor complexes in the lung and intestinal tract is restricted to epithelial cells and a few other, undefined cell types. Interestingly, we found that SARS coronavirus was present in feces from infected mice lacking receptors for both type I and type III IFN but not in those from mice lacking single receptors, supporting the view that IFN-lambda contributes to the control of viral infections in epithelial cells of both respiratory and gastrointestinal tracts.

It has been shown that all three classes of interferons enhance the expression of the major histocompatibility class I antigens (HLA-A,B,C;H-2) on a wide variety of cell types (1-10). However, their effect on the expression of the class II antigens (HLA-DR, Ia), which play a major part in cellular interactions that initiate an immune response, is more controversial. The predominate findings have been that the interferons specifically increase the synthesis and expression of only the class I antigens (3, 4, 6, 8, 10, 11). We report here that recombinant interferon-gamma (IFN-gamma) increases the synthesis and expression of the HLA-DR (la-like) antigens as well as beta 2-microglobulin (beta 2-m), a low m.w. subunit of HLA, on human melanoma cells. No increase in HLA-DR was detected on these melanoma cells with leukocyte interferon (IFN-alpha) at doses 400 times higher than the maximum dose of IFN-gamma. These findings were extended to show that pure IFN-gamma also increases the expression of the HLA-DR antigens on normal peripheral blood monocytes, whereas recombinant IFN-alpha at a similar dose had little effect on the expression of this surface antigen. These findings suggest a specialized role for IFN-gamma in immune regulation in comparison with IFN-alpha.

The role of bacteria in the initiation of periodontitis is well-documented and the end result, destruction of the alveolar bone and periodontal connective tissue, is readily observed; but the events occurring between these two points in time remain obscure and are the focus of this paper. Bacteria induce tissue destruction indirectly by activating host defense cells, which in turn produce and release mediators that stimulate the effectors of connective tissue breakdown. Components of microbial plaque have the capacity to induce the initial infiltrate of inflammatory cells including lymphocytes, macrophages, and PMNs. Microbial components, especially lipopolysaccharide (LPS), have the capacity to activate macrophages to synthesize and secrete a wide array of molecules including the cytokines interleukin-1 (IL-1) and tumor-necrosis factor-alpha (TNF-alpha), prostaglandins, especially PGE2, and hydrolytic enzymes. Likewise, bacterial substances activate T lymphocytes and they produce IL-1 and lymphotoxin (LT), a molecule having properties very similar to TNF-alpha. These cytokines manifest potent proinflammatory and catabolic activities, and play key roles in periodontal tissue breakdown. They induce fibroblasts and macrophages to produce neutral metalloproteinases such as procollagenase and prostromelysin, the serine proteinase urokinase-type plasminogen activator (u-PA), tissue inhibitor of metalloproteinase (TIMP), and prostaglandins, u-PA converts plasminogen into plasmin, which can activate neutral metalloproteinase proenzymes, and these enzymes degrade the extracellular matrix components. TIMP inactivates the active enzymes and thereby blocks further tissue degradation. Several amplification and suppression mechanisms are involved in the process. While LPS activates macrophages to produce IL-1, IL-1 is autostimulatory and can therefore amplify and perpetuate its own production. Interferon-gamma (INF-gamma) suppresses autostimulation, but it enhances LPS-induced IL-1 production. PGE2 exerts a control over the whole process by suppressing production of both IL-1 and TNF-alpha. Furthermore, the activated cells produce an IL-1 receptor antagonist that binds to the IL-1 receptor but does not induce the biologic consequences of IL-1 binding. Other cytokines such as transforming growth factor-beta (TGF-beta) suppress production of metalloproteinases and u-PA. Thus the progression and extent of tissue degradation is likely to be determined in major part by relative concentrations and half-life of IL-1, TNF-alpha, and related cytokines, competing molecules such as the IL-1 receptor antagonist, and suppressive molecules such as TGF-beta and PGE2. These molecules control levels of latent and active metalloproteinase and u-PA, and the availability and concentration of TIMP determines the extent and duration of degradative activity.

Actin is abundant in the nucleus and has been implicated in transcription; however, the nature of this involvement has not been established. Here we demonstrate that beta-actin is critically involved in transcription because antibodies directed against beta-actin, but not muscle actin, inhibited transcription in vivo and in vitro. Chromatin immunoprecipitation assays demonstrated the recruitment of actin to the promoter region of the interferon-gamma-inducible MHC2TA gene as well as the interferon-alpha-inducible G1P3 gene. Further investigation revealed that actin and RNA polymerase II co-localize in vivo and also co-purify. We employed an in vitro system with purified nuclear components to demonstrate that antibodies to beta-actin block the initiation of transcription. This assay also demonstrates that beta-actin stimulates transcription by RNA polymerase II. Finally, DNA-binding experiments established the presence of beta-actin in pre-initiation complexes and also showed that the depletion of actin prevented the formation of pre-initiation complexes. Together, these data suggest a fundamental role for actin in the initiation of transcription by RNA polymerase II.

Dengue virus (DEN) causes dengue fever and dengue hemorrhagic fever/dengue shock syndrome, which are major public health problems worldwide. The immune factors that control DEN infection or contribute to severe disease are neither well understood nor easy to examine in humans. In this study, we used wild-type and congenic mice lacking various components of the immune system to study the immune mechanisms in the response to DEN infection. Our results demonstrate that alpha/beta interferon (IFN-alpha/beta) and IFN-gamma receptors have critical, nonoverlapping functions in resolving primary DEN infection. Furthermore, we show that IFN-alpha/beta receptor-mediated action limits initial DEN replication in extraneural sites and controls subsequent viral spread into the central nervous system (CNS). In contrast, IFN-gamma receptor-mediated responses seem to act at later stages of DEN disease by restricting viral replication in the periphery and eliminating virus from the CNS. Mice deficient in B, CD4(+) T, or CD8(+) T cells had no increased susceptibility to DEN; however, RAG mice (deficient in both B and T cells) were partially susceptible to DEN infection. In summary, (i) IFN-alpha/beta is critical for early immune responses to DEN infection, (ii) IFN-gamma-mediated immune responses are crucial for both early and late clearance of DEN infection in mice, and (iii) the IFN system plays a more important role than T- and B-cell-dependent immunity in resistance to primary DEN infection in mice.

The interferon (IFN) response is the first line of defense against viral infections, and the majority of viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile viruses, the New York 99 strain (NY99) and Kunjin virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-alpha/beta) and gamma IFN (IFN-gamma) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-alpha. IFN-alpha signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins responsible for this inhibition.

Hepatitis B virus (HBV) causes chronic infection in more than 350 million people worldwide. It replicates in hepatocytes but is non-cytopathic; liver damage is thought to be immune mediated. Here, we investigated the role of innate immune responses in mediating liver damage in patients with chronic HBV infection. Longitudinal analysis revealed a temporal correlation between flares of liver inflammation and fluctuations in interleukin (IL)-8, interferon (IFN)-alpha, and natural killer (NK) cell expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) directly ex vivo. A cross-sectional study confirmed these findings in patients with HBV-related liver inflammation compared with healthy carriers. Activated, TRAIL-expressing NK cells were further enriched in the liver of patients with chronic HBV infection, while their hepatocytes expressed increased levels of a TRAIL death-inducing receptor. IFN-alpha concentrations found in patients were capable of activating NK cells to induce TRAIL-mediated hepatocyte apoptosis in vitro. The pathogenic potential of this pathway could be further enhanced by the ability of the IFN-alpha/IL-8 combination to dysregulate the balance of death-inducing and regulatory TRAIL receptors expressed on hepatocytes. We conclude that NK cells may contribute to liver inflammation by TRAIL-mediated death of hepatocytes and demonstrate that this non-antigen-specific mechanism can be switched on by cytokines produced during active HBV infection.

Destruction of li by proteolysis is required for MHC class II molecules to bind antigenic peptides, and for transport of the resulting complexes to the cell surface. The cysteine protease cathepsin S is highly expressed in spleen, lymphocytes, monocytes, and other class II-positive cells, and is inducible with interferon-gamma. Specific inhibition of cathepsin S in B lymphoblastoid cells prevented complete proteolysis of li, resulting in accumulation of a class II-associated 13 kDa li fragment in vivo. Consequently, the formation of SDS-stable complexes was markedly reduced. Purified cathepsin S, but not cathepsin B, H, or D, specifically digested li from alpha beta li trimers, generating alpha beta-CLIP complexes capable of binding exogenously added peptide in vitro. Thus, cathepsin S is essential in B cells for effective li proteolysis necessary to render class II molecules competent for binding peptides.

MxA and MxB are interferon-induced proteins of human cells and are related to the murine protein Mx1, which confers selective resistance to influenza virus. In contrast to the nuclear murine protein Mx1, MxA and MxB are located in the cytoplasm, and their role in the interferon-induced antiviral state was unknown. In this report we show that transfected cell lines expressing MxA acquired a high degree of resistance to influenza A virus. Surprisingly, MxA also conferred resistance to vesicular stomatitis virus. Expression of MxA in transfected 3T3 cells had no effect on the multiplication of two picornaviruses, a togavirus, or herpes simplex virus type 1. Treatment of MxA-expressing cells with antibodies to mouse alpha-beta interferon did not abolish the resistance phenotype. The conclusion that resistance to influenza virus and vesicular stomatitis virus was due to the specific action of MxA is further supported by the observation that transfected 3T3 cell lines expressing the related MxB failed to acquire virus resistance.

The presence and the role of interleukin 10 (IL-10), a potent cytokine synthesis inhibitory factor and antiinflammatory cytokine, were investigated in rheumatoid arthritis (RA). The expression of both mRNA and protein for IL-10 could be demonstrated in RA and osteoarthritis (OA) joints. Human IL-10 mRNA could be demonstrated by polymerase chain reaction amplification of cDNA made by reverse transcription of total RNA extracted directly from synovial tissue in five out of five RA and four out of five OA patients. IL-10 protein was demonstrated by specific immunoassay and immunohistology. IL-10 protein was spontaneously produced in all 11 RA and 17 OA synovial membrane cultures investigated, and this production was sustained for up to 5 d in culture in the absence of any extrinsic stimulation. IL-10 protein could also be detected by immunohistology in all five RA and four OA synovial membrane biopsies investigated, but not three normal synovial membranes. Immunohistology revealed that the IL-10 was localized to the synovial membrane lining layer and mononuclear cell aggregates. Immunofluorescence double staining revealed that the sources of IL-10 were monocytes in the lining layer, and T cells in the mononuclear cell aggregates. We found evidence that the IL-10 expression was functionally relevant, as neutralization of endogenously produced IL-10 in the RA synovial membrane cultures resulted in a two- to threefold increase in the protein levels of proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and IL-1 beta, although IL-6 and IL-8 levels were not affected. The addition of exogenous recombinant IL-10 to the RA synovial membrane cultures resulted in a two- to threefold decrease in the levels of TNF-alpha and IL-1 beta. IL-8 levels were reduced by day 5; however, IL-6 levels were not affected by exogenous IL-10. Neutralization of the endogenous IL-10 in two out of seven RA synovial membrane cultures resulted in the expression of detectable levels of interferon gamma (561-1,050 pg/ml). Taken together, the above findings suggest that IL-10 is spontaneously produced in RA and OA and is an important immunoregulatory component in the cytokine network of RA, regulating monocyte and in some cases T cell cytokine production.

Interferons (IFN) alpha/beta and gamma induce the formation of two transcriptional activators: gamma-activating factor (GAF) and interferon-stimulated gamma factor 3 (ISGF3). We report a natural heterozygous germline STAT1 mutation associated with susceptibility to mycobacterial but not viral disease. This mutation causes a loss of GAF and ISGF3 activation but is dominant for one cellular phenotype and recessive for the other. It impairs the nuclear accumulation of GAF but not of ISGF3 in heterozygous cells stimulated by IFNs. Thus, the antimycobacterial, but not the antiviral, effects of human IFNs are principally mediated by GAF.

The influenza A virus NS1 protein (NS1A protein) binds and inhibits the function of the 30-kDa subunit of CPSF, a cellular factor that is required for the 3'-end processing of cellular pre-mRNAs. Here we generate a recombinant influenza A/Udorn/72 virus that encodes an NS1A protein containing a mutated binding site for the 30-kDa subunit of CPSF. This mutant virus is substantially attenuated, indicating that this binding site in the NS1A protein is required for efficient virus replication. Using this mutant virus, we show that NS1A binding to CPSF mediates the viral posttranscriptional countermeasure against the initial cellular antiviral response--the interferon-alpha/beta (IFN-alpha/beta)-independent activation of the transcription of cellular antiviral genes, which requires the interferon regulatory factor-3 (IRF-3) transcription factor that is activated by virus infection. Whereas the posttranscriptional processing of these cellular antiviral pre-mRNAs is inhibited in cells infected by wild-type influenza A virus, functional antiviral mRNAs are produced in cells infected by the mutant virus. These results establish that the binding of 30-kDa CPSF to the NS1A protein is largely responsible for the posttranscriptional inhibition of the processing of these cellular antiviral pre-mRNAs. Mutation of this binding site in the NS1A protein also affects a second cellular antiviral response: in cells infected by the mutant virus, IFN-beta mRNA is produced earlier and in larger amounts.

Type I (alpha, beta) and type II (gamma) interferons (IFNs) can restrict the growth of many cell types. INF-stimulated gene transcription, a key early event in IFN response, acts through the Janus kinase-signal transducers and activators of transcription pathway, in which both IFN-alpha and IFN-gamma activate the transcription factor Stat1. A cell line lacking Stat1 (U3A) was not growth-arrested by IFN-alpha or IFN-gamma, and experiments were carried out with U3A cells permanently expressing normal or various mutant forms of Stat1 protein. Only cells in which complete Stat1 activity was available (Stat1alpha) were growth-inhibited by IFN-gamma. A mutant that supports 20-30% normal transcription did not cause growth restraint. In contrast, IFN-alpha growth restraint was imposed by cells producing Stat1beta, which lacks transcriptional activation potential. This parallels earlier results showing the truncated Stat1 can function in IFN-alpha gene activation. In addition to experiments on long-term cultured cells, we also found that wild-type primary mouse embryonic fibroblasts were inhibited by IFNs, but fibroblasts from Stat1-deficient mouse embryos were not inhibited by IFNs.

Lymphopenia and increasing viral load in the first 10 days of severe acute respiratory syndrome (SARS) suggested immune evasion by SARS-coronavirus (CoV). In this study, we focused on dendritic cells (DCs) which play important roles in linking the innate and adaptive immunity. SARS-CoV was shown to infect both immature and mature human monocyte-derived DCs by electron microscopy and immunofluorescence. The detection of negative strands of SARS-CoV RNA in DCs suggested viral replication. However, no increase in viral RNA was observed. Using cytopathic assays, no increase in virus titer was detected in infected DCs and cell-culture supernatant, confirming that virus replication was incomplete. No induction of apoptosis or maturation was detected in SARS-CoV-infected DCs. The SARS-CoV-infected DCs showed low expression of antiviral cytokines (interferon alpha [IFN-alpha], IFN-beta, IFN-gamma, and interleukin 12p40 [IL-12p40]), moderate up-regulation of proinflammatory cytokines (tumor necrosis factor alpha [TNF-alpha] and IL-6) but significant up-regulation of inflammatory chemokines (macrophage inflammatory protein 1alpha [MIP-1alpha], regulated on activation normal T cell expressed and secreted [RANTES]), interferon-inducible protein of 10 kDa [IP-10], and monocyte chemoattractant protein 1 [MCP-1]). The lack of antiviral cytokine response against a background of intense chemokine up-regulation could represent a mechanism of immune evasion by SARS-CoV.