In order to identify novel mediators synthesized in activated macrophages, a cDNA library was prepared from cultures of the mouse macrophage cell line RAW 264.7 that had been treated with lymphokine-rich conditioned medium from mitogen-stimulated mouse spleen cells. Differential plaque hybridization identified a cDNA, designated m119, that detected a 1.6-kilobase mRNA that accumulated in response to gamma-interferon (IFN-gamma) but not in response to other macrophage activators, including IFN-alpha, IFN-beta, and lipopolysaccharide. The mRNA encoded a predicted protein of Mr 14,461 containing a 21-amino acid signal peptide. The primary structure of the predicted protein indicated that it is a member of a recently described family of cytokines related to platelet factor 4, including Gro/melanoma growth stimulatory activity and neutrophil-activating peptide/interleukin 8. The selective induction of the m119 mRNA by IFN-gamma that the predicted m119 protein mediates a macrophage activity regulated by IFN-gamma. The m119 protein may be a cytokine that affects the growth, movement, or activation state of cells that participate in immune and inflammatory responses. It is proposed that the gene encoding this protein be called mig, for monokine induced by gamma interferon.

Germline mutations in five autosomal genes involved in interleukin (IL)-12-dependent, interferon (IFN)-gamma-mediated immunity cause Mendelian susceptibility to mycobacterial diseases (MSMD). The molecular basis of X-linked recessive (XR)-MSMD remains unknown. We report here mutations in the leucine zipper (LZ) domain of the NF-kappaB essential modulator (NEMO) gene in three unrelated kindreds with XR-MSMD. The mutant proteins were produced in normal amounts in blood and fibroblastic cells. However, the patients' monocytes presented an intrinsic defect in T cell-dependent IL-12 production, resulting in defective IFN-gamma secretion by T cells. IL-12 production was also impaired as the result of a specific defect in NEMO- and NF-kappaB/c-Rel-mediated CD40 signaling after the stimulation of monocytes and dendritic cells by CD40L-expressing T cells and fibroblasts, respectively. However, the CD40-dependent up-regulation of costimulatory molecules of dendritic cells and the proliferation and immunoglobulin class switch of B cells were normal. Moreover, the patients' blood and fibroblastic cells responded to other NF-kappaB activators, such as tumor necrosis factor-alpha, IL-1beta, and lipopolysaccharide. These two mutations in the NEMO LZ domain provide the first genetic etiology of XR-MSMD. They also demonstrate the importance of the T cell- and CD40L-triggered, CD40-, and NEMO/NF-kappaB/c-Rel-mediated induction of IL-12 by monocyte-derived cells for protective immunity to mycobacteria in humans.

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever and neutrophil-mediated serosal inflammation. We recently identified the gene causing FMF, designated MEFV, and found it to be expressed in mature neutrophils, suggesting that it functions as an inflammatory regulator. To facilitate our understanding of the normal function of MEFV, we extended our previous studies. MEFV messenger RNA was detected by reverse transcriptase-polymerase chain reaction in bone marrow leukocytes, with differential expression observed among cells by in situ hybridization. CD34 hematopoietic stem-cell cultures induced toward the granulocytic lineage expressed MEFV at the myelocyte stage, concurrently with lineage commitment. The prepromyelocytic cell line HL60 expressed MEFV only at granulocytic and monocytic differentiation. MEFV was also expressed in the monocytic cell lines U937 and THP-1. Among peripheral blood leukocytes, MEFV expression was detected in neutrophils, eosinophils, and to varying degrees, monocytes. Consistent with the tissue specificity of expression, complete sequencing and analysis of upstream regulatory regions of MEFV revealed homology to myeloid-specific promoters and to more broadly expressed inflammatory promoter elements. In vitro stimulation of monocytes with the proinflammatory agents interferon (IFN) gamma, tumor necrosis factor, and lipopolysaccharide induced MEFV expression, whereas the antiinflammatory cytokines interleukin (IL) 4, IL-10, and transforming growth factor beta inhibited such expression. Induction by IFN-gamma occurred rapidly and was resistant to cycloheximide. IFN-alpha also induced MEFV expression. In granulocytes, MEFV was up-regulated by IFN-gamma and the combination of IFN-alpha and colchicine. These results refine understanding of MEFV by placing the gene in the myelomonocytic-specific proinflammatory pathway and identifying it as an IFN-gamma immediate early gene.

MoPn-specific T-cell clones were isolated from a T-cell line that was capable of curing chlamydial genital infection by the Chlamydia trachomatis agent of mouse pneumonitis (MoPn) after adoptive transfer. Two clones (designated as 2.14-0 and 2.14-3) were characterized by flow cytometry techniques to be homogenous for L3T4, CD3, and alpha/beta T cell receptor (TcR) T-helper cell markers. The two clones were biovar specific, because they reacted to MoPn but not the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis (GPIC) or C. trachomatis, serovar type E. Cytokine profile analysis, by a combination of bioassays, ELISA, and slot/Northern blotting for specific cytokine messenger RNAs, further revealed that cultures of antigen-stimulated clone 2.14-0 contained interleukin-2 (IL-2), tumor necrosis factor-alpha, and gamma interferon (a T helper 1 cell [Th1] profile). Clone 2.14-3 was also positive for gamma interferon, a level much lower than that of clone 2.14-0, and negative for IL-4 secretion, suggesting a Th1 profile as well. The ability of these clones to bring about the resolution of the chronic genital chlamydial infection of nude mice was tested by the adoptive transfer of 10(7) cells of each clone into the mice. By 4 weeks after cell transfer of clone 2.14-0, 81% of recipient nude mice (30 of 37) resolved the disease. In contrast, clone 2.14-3 or a control T-cell clone specific for a heterologous antigen were unable to resolve the infection in 20 recipients in each case, even after 100 days.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND

Mosquito-borne Zika virus (ZIKV) typically causes a mild and self-limiting illness known as Zika fever, which often is accompanied by maculopapular rash, headache, and myalgia. During the current outbreak in South America, ZIKV infection during pregnancy has been hypothesized to cause microcephaly and other diseases. The detection of ZIKV in fetal brain tissue supports this hypothesis. Because human infections with ZIKV historically have remained sporadic and, until recently, have been limited to small-scale epidemics, neither the disease caused by ZIKV nor the molecular determinants of virulence and/or pathogenicity have been well characterized. Here, we describe a small animal model for wild-type ZIKV of the Asian lineage.

RESULTS

Using mice deficient in interferon α/β and Ɣ receptors (AG129 mice), we report that these animals were highly susceptible to ZIKV infection and disease, succumbing within seven to eight days. Rapid viremic dissemination was observed in visceral organs and brain; but only was associated with severe pathologies in the brain and muscle. Finally, these results were consistent across challenge routes, age of mice, and inoculum doses. These data represent a mouse model for ZIKV that is not dependent on adapting ZIKV to intracerebral passage in mice.

CONCLUSIONS

Foot pad injection of AG129 mice with ZIKV represents a biologically relevant model for studying ZIKV infection and disease development following wild-type virus inoculation without the requirement for adaptation of the virus or intracerebral delivery of the virus. This newly developed Zika disease model can be exploited to identify determinants of ZIKV virulence and reveal molecular mechanisms that control the virus-host interaction, providing a framework for rational design of acute phase therapeutics and for vaccine efficacy testing.

T-cell-based immunotherapies provide a promising means of cancer treatment although durable antitumor responses are infrequent. A potential reason for these shortcomings may lie in the observed lack of trafficking of specific T cells to tumor. Our increasing knowledge of the process of trafficking involving adhesion molecules and chemokines affords us the opportunity to intervene and correct deficiencies in this process. Chemokines can be expressed by a range of tumors and may serve as suitable targets for directing specific T cells toward tumor. We initially sought to identify which chemokines were produced by a range of human tumor cell lines, and which chemokines and chemokine receptors were expressed by cultured T cells. We identified two chemokines: Growth-Regulated Oncogene-alpha (Gro-alpha; CXCL1) and Regulated on Activation Normal T Cell-Expressed and Secreted (RANTES; CCL5), to be secreted by several human tumor cell lines. Expression was also detected in fine-needle aspirates of melanoma from patients. In addition, we determined the expression of several chemokine receptors on cultured human T cells including CCR1, CCR2, CCR4, CCR5, CXCR3, and CXCR4. Cultured, activated human T cells expressed the chemokines lymphotactin (XCL1), RANTES, macrophage inflammatory protein-1 alpha (MIP-1 alpha; CCL3) and MIP-1 beta (CCL4), but no appreciable Gro-alpha. In a strategy to direct T cells toward chemokines expressed by tumors we chose Gro-alpha as the target chemokine because it was produced by tumor and not by T cells themselves. However, T cells did not express the receptor for Gro-alpha, CXCR2, and therefore, T cells were transduced with a retroviral vector encoding CXCR2. Calcium ion mobilization, an important first step in chemokine receptor signaling, was subsequently demonstrated in transduced T cells in response to Gro-alpha. In addition, Gro-alpha was chemotactic for T cells expressing CXCR2 in vitro toward both recombinant protein and tumor-derived chemokine. Interestingly we demonstrate, for the first time, that Gro-alpha was able to induce interferon-gamma (IFN-gamma) secretion from transduced T cells, thereby extending our knowledge of other potential functions of CXCR2. This study demonstrates the feasibility of redirecting the migration properties of T cells toward chemokines secreted by tumors.

Of 37 pancreases removed at necropsy from patients with type 1 diabetes 34 had residual beta cells. In 33 of the 34 the beta cells were positive for immunoreactive alpha-interferon, and this finding in islets was related to hyperexpression of class I major histocompatibility complex (MHC) antigens. Of islets showing class I hyperexpression 93% contained alpha-interferon compared with 0.4% of those showing no hyperexpression. Among 80 control pancreases significant numbers of beta cells containing alpha-interferon were present only in 4 cases of acute infantile viral pancreatitis, known to be caused by Coxsackie-B viral infection in 3 cases. Chronic viral infection of beta cells may underlie the pathogenesis of some cases of type 1 diabetes.

The genes for the alpha and beta chains of a highly reactive anti-MART-1 T-cell receptor were isolated from T-lymphocytes that mediated in vivo regression of tumor in a patient with metastatic melanoma. These genes were cloned and inserted into MSCV-based retroviral vectors. After transduction, greater than 50% gene transfer efficiency was demonstrated in primary T-lymphocytes stimulated by an anti-CD3 antibody. The specificity and biologic activity of TCR gene-transduced T-cells was determined by cytokine production after coculture of T-cells with stimulator cells pulsed with MART-1 peptide. The production of interferon-gamma and granulocyte macrophage-colony stimulating factor (GM-CSF) was comparable to highly active MART-1 specific peripheral blood lymphocytes (PBL) in the amount of cytokine produced and transduced cells recognized peptide pulsed cells at dilutions similar to cytotoxic T lymphocyte (CTL) clones. Human leukocyte antigen (HLA) class I restricted recognition was demonstrated by mobilization of degranulation marker CD107a, by cell lysis, by cytokine production, and by proliferation in the presence of HLA-A2-positive but not HLA-A2-negative melanoma cell lines. Similar data was obtained when tumor-infiltrating lymphocytes (TIL) were transduced with the TCR genes, converting previously nonreactive cells to tumor reactive cells. TCR-transduced T-cells are thus attractive candidates for evaluation in cell transfer therapies of patients with cancer.

Protein ISGylation is unique among ubiquitin-like conjugation systems in that the expression and conjugation processes are induced by specific stimuli, mainly via the alpha/beta interferon signaling pathway. It has been suggested that protein ISGylation plays a special role in the immune response, because of its interferon-signal dependency and its appearance only in higher eukaryotic organisms. Here, we report the identification of an ISG15-conjugating enzyme, Ubc8. Like other components of the protein ISGylation system (ISG15, UBE1L, and UBP43), Ubc8 is an interferon-inducible protein. Ubc8 clearly mediates protein ISGylation in transfection assays. The reduction of Ubc8 expression by small interfering RNA causes a decrease in protein ISGylation in HeLa cells upon interferon treatment. Neither UbcH7/UbcM4, the closest homologue of Ubc8 among known ubiquitin E2s, nor the small ubiquitin-like modifier E2 Ubc9 supports protein ISGylation. These findings strongly suggest that Ubc8 is a major ISG15-conjugating enzyme responsible for protein ISGylation upon interferon stimulation. Furthermore, we established an assay system to detect ISGylated target proteins by cotransfection of ISG15, UBE1L, and Ubc8 together with a target protein to be analyzed. This method provides an easy and effective way to identify new targets for the ISGylation system and will facilitate related studies.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells that has the ability to induce gamma interferon (IFN-gamma) secretion by T and natural killer cells and to generate normal Th1 responses. These properties suggest that IL-12 may play an important role in the immune response to many viruses, including hepatitis B virus (HBV). Recently, we have shown that HBV-specific cytotoxic T lymphocytes inhibit HBV replication in the livers of transgenic mice by a noncytolytic process that is mediated in part by IFN-gamma. In the current study, we demonstrated that the same antiviral response can be initiated by recombinant murine IL-12 and we showed that the antiviral effect of IL-12 extends to extrahepatic sites such as the kidney. Southern blot analyses revealed the complete disappearance of HBV replicative intermediates from liver and kidney tissues at IL-12 doses that induce little or no inflammation in these tissues. In addition, immunohistochemical analysis demonstrated the disappearance of cytoplasmic hepatitis B core antigen from both tissues after IL-12 treatment, suggesting that IL-12 either prevents the assembly or triggers the degradation of the nucleocapsid particles within which HBV replication occurs. Importantly, we demonstrated that although IFN-gamma, tumor necrosis factor alpha, and IFN-alpha/beta mRNA are induced in the liver and kidney after IL-12 administration, the antiviral effect of IL-12 is mediated principally by its ability to induce IFN-gamma production in this model. These results suggest that IL-12, through its ability to induce IFN-gamma, probably plays an important role in the antiviral immune response to HBV during natural infection. Further, since relatively nontoxic doses of recombinant IL-12 profoundly inhibit HBV replication in the liver and extrahepatic sites in this model, IL-12 may have therapeutic value as an antiviral agent for the treatment of chronic HBV infection.

Induction of high systemic levels of type 1 interferons (IFNs) IFN-alpha and IFN-beta is a hallmark of many viral infections. In addition to their potent antiviral effects, these cytokines mediate a number of immunoregulatory functions and can promote IFN-gamma expression in T cells. However, during viral infections of mice IFN-gamma production is not always observed at the same time as systemic IFN-alpha/beta production and when, elicited at these times, is IFN-alpha/beta-independent. We demonstrate that type 1 interferons not only fail to induce, but also act to inhibit, IFN-gamma expression by both NK and T cells. The mechanism of inhibition is dependent upon the IFN-alpha/beta receptor and the signal transducer and activator of transcription 1 (STAT1). In the absence of STAT1, not only are the IFN-alpha/beta-mediated inhibitory effects completely abrogated, but the cytokines themselves can induce IFN-gamma expression. These results indicate that endogenous biochemical pathways are in place to negatively regulate NK and T cell IFN-gamma expression elicited by IFN-alpha/beta or other stimuli, at times of innate responses to viral infections. They also show that type 1 interferon signaling can occur through STAT1-dependent and independent mechanisms and suggest that efficient induction of IFN-gamma expression by IFN-alpha/beta requires STAT1 regulation. Such immunoregulatory pathways may be critical for shaping the endogenous innate and virus-specific adaptive immune responses to viral infections.

Purified peripheral murine T cells, in the presence of concanavalin A, can be activated to produce interleukin 2 (IL-2) through stimulation either with a previously described murine lymphokine designated T cell-activating factor (TAF) or with a cloned human lymphokine that has been called beta 2 interferon, B-cell-stimulatory factor 2, hybridoma growth factor, inducible 26-kDa protein, or hematopoietic colony-stimulating factor 309 by different investigators. We and others propose the designation interleukin 6 (IL-6) for the latter molecule. Our experiments demonstrate that either murine TAF or human IL-6 can restore the ability of purified T cells to proliferate in response to Con A or antibodies against the T-cell antigen receptor. Most if not all of the proliferation can be blocked by antibodies against the alpha chain of the IL-2 receptor. Furthermore, highly purified CD8- T cells can be activated by IL-6 in the presence of Con A to secrete IL-2. We propose that IL-6 and murine TAF are important "second signals" in primary antigen-receptor-dependent T-cell activation. Whether or not murine TAF is a homologue of human IL-6 remains to be determined.

Ebola virus (EBOV) infections are characterized by dysregulation of normal host immune responses. Insight into the mechanism came from recent studies in nonhuman primates, which showed that EBOV infects cells of the mononuclear phagocyte system (MPS), resulting in apoptosis of bystander lymphocytes. In this study, we evaluated serum levels of cytokines/chemokines in EBOV-infected nonhuman primates, as possible correlates of this bystander apoptosis. Increased levels of interferon (IFN)-alpha, IFN-beta, interleukin (IL)-6, IL-18, MIP-1alpha, and MIP-1beta were observed in all EBOV-infected monkeys, indicating the occurrence of a strong proinflammatory response. To investigate the mechanism(s) involved in lymphoid apoptosis, soluble Fas (sFas) and nitrate accumulation were measured. sFas was detected in 4/9 animals, while, elevations of nitrate accumulation occurred in 3/3 animals. To further evaluate the potential role of these factors in the observed bystander apoptosis and intact animals, in vitro cultures were prepared of adherent human monocytes/macrophages (PHM), and monocytes differentiated into immature dendritic cells (DC). These cultures were infected with EBOV and analyzed for cytokine/chemokine induction and expression of apoptosis-related genes. In addition, the in vitro EBOV infection of peripheral blood mononuclear cells (PBMC) resulted in strong cytokine/chemokine induction, a marked increase in lactate dehydrogenase (LDH) activity, and an increase in the number of apoptotic lymphocytes examined by electron microscopy. Increased levels of sFAS were detected in PHM cultures, although, <10% of the cells were positive by immunohistochemistry. In contrast, >90% of EBOV-infected PHM were positive for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by immunohistochemistry, RNA analysis, and flow cytometry. Inactivated EBOV also effected increased TRAIL expression in PHM, suggesting that the TNF receptor superfamily may be involved in apoptosis of the host lymphoid cells, and that induction may occur independent of viral replication. In further studies with infected PHM, expression of MHC II was remarkably suppressed after 6 days, an additional correlate of immunological dysregulation. In conclusion, our findings suggest that infection of mononuclear phagocytes is critical, triggering a cascade of events involving cytokines/chemokines and oxygen free radicals. It is the consequence of these events rather than direct viral infection that results in much of the observed pathology. Identification of cytokine/chemokine, nitric oxide, and reactive oxygen species involvement in the observed filoviral pathogenesis may lend insight into the rational design of therapeutic countermeasures of filoviral pathogenesis.

The interferons (IFN) are one of the body's natural defensive responses to such foreign components as microbes, tumors, and antigens. The IFN response begins with the production of the IFN proteins (alpha, beta, and gamma), which then induce the antiviral, antimicrobial, antitumor, and immunomodulatory actions of IFN. Recent advances have led to Food and Drug Administration approval of five clinical indications for IFN. Interferon alfa is approved for hairy-cell leukemia, condyloma acuminatum, Kaposi's sarcoma in the acquired immunodeficiency syndrome, and non-A, non-B (type C) viral hepatitis. Interferon gamma has properties distinctive from those of IFNs alpha and beta and is approved as an immunomodulatory treatment for chronic granulomatous disease. Promising clinical results with IFNs have also been reported for basal cell carcinoma, chronic myelogenous leukemia, cutaneous squamous cell carcinoma, early human immunodeficiency virus infection, hepatitis B, and laryngeal papillomatosis. Future clinical uses of IFNs may emphasize combination therapy with other cytokines, chemotherapy, radiation, surgery, hyperthermia, or hormones.

Enteroviruses are believed to contribute to the pathogenesis of type 1 diabetes mellitus (T1DM). In this Review, the interplay between infection with enteroviruses, the immune system and host genes is discussed. Data from retrospective and prospective epidemiological studies strongly suggest the involvement of enteroviruses, such as coxsackievirus B, in the development of T1DM. Enteroviral RNA and/or proteins can be detected in tissues of patients with T1DM. Isolation of coxsackievirus Bbeta cells and the activation of innate immunity and inflammation. In contrast with its antiviral role, virus-induced interferon alpha can be deleterious, acting as an initiator of the autoimmunity directed against beta cells. Enteroviruses, through persistent and/or successive infections, can interact with the adaptive immune system. Host genes, such as IFIH1, that influence susceptibility to T1DM are associated with antiviral activities. An increased activity of the IFIH1 protein may promote the development of T1DM. An improved knowledge of the pathogenic mechanisms of enterovirus infections should help to uncover preventive strategies for T1DM.

BACKGROUND

The autoimmune regulator (AIRE) gene influences thymic self-tolerance induction. In autoimmune polyendocrinopathy syndrome type 1 (APS1; OMIM 240300), recessive AIRE mutations lead to autoimmunity targetting endocrine and other epithelial tissues, although chronic candidiasis usually appears first. Autoimmunity and chronic candidiasis can associate with thymomas as well. Patients with these tumours frequently also have high titre immunoglobulin G autoantibodies neutralising type I interferon (IFN)-alpha and IFN-omega, which are secreted signalling proteins of the cytokine superfamily involved in both innate and adaptive immunity.

RESULTS

We tested for serum autoantibodies to type I IFNs and other immunoregulatory cytokines using specific binding and neutralisation assays. Unexpectedly, in 60/60 Finnish and 16/16 Norwegian APS1 patients with both AIRE alleles mutated, we found high titre neutralising immunoglobulin G autoantibodies to most IFN-<em>alpha</em> subtypes and especially IFN-omega (60% homologous to IFN-<em>alpha</em>)-mostly in the earliest samples. We found lower titres against IFN-beta (30% homologous to IFN-<em>alpha</em>) in 23% of patients; two-thirds of these (from Finland only) also had low titres against the distantly related "type III IFN" (IFN-lambda1; alias interleukin-29). However, autoantibodies to the unrelated type II IFN, IFN-gamma, and other immunoregulatory cytokines, such as interleukin-10 and interleukin-12, were much rarer and did not neutralise. Neutralising titres against type I IFNs averaged even higher in patients with APS1 than in patients with thymomas. Anti-type I IFN autoantibodies preceded overt candidiasis (and several of the autoimmune disorders) in the informative patients, and persisted for decades thereafter. They were undetectable in unaffected heterozygous relatives of APS1 probands (except for low titres against IFN-lambda1), in APS2 patients, and in isolated cases of the endocrine diseases most typical of APS1, so they appear to be APS1-specific. Looking for potentially autoimmunising cell types, we found numerous IFN-<em>alpha</em>(+) antigen-presenting cells-plus strong evidence of local IFN secretion-in the normal thymic medulla (where AIRE expression is strongest), and also in normal germinal centres, where it could perpetuate these autoantibody responses once initiated. IFN-<em>alpha</em>2 and IFN-<em>alpha</em>8 transcripts were also more abundant in antigen-presenting cells cultured from an APS1 patient's blood than from age-matched healthy controls.

CONCLUSIONS

These apparently spontaneous autoantibody responses to IFNs, particularly IFN-<em>alpha</em> and IFN-omega, segregate like a recessive trait; their high "penetrance" is especially remarkable for such a variable condition. Their apparent restriction to APS1 patients implies practical value in the clinic, e.g., in diagnosing unusual or prodromal AIRE-mutant patients with only single components of APS1, and possibly in prognosis if they prove to predict its onset. These autoantibody responses also raise numerous questions, e.g., about the rarity of other infections in APS1. Moreover, there must also be clues to autoimmunising mechanisms/cell types in the hierarchy of preferences for IFN-omega, IFN-<em>alpha</em>8, IFN-<em>alpha</em>2, and IFN-beta and IFN-lambda1.

Vertebrate innate immunity provides a first line of defence against pathogens such as viruses and bacteria. Viral infection activates a potent innate immune response, which can be triggered by double-stranded (ds)RNA produced during viral replication. Here, we report that mammalian cells lacking the death-domain-containing protein FADD are defective in intracellular dsRNA-activated gene expression, including production of type I (alpha/beta) interferons, and are thus very susceptible to viral infection. The signalling pathway incorporating FADD is largely independent of Toll-like receptor 3 and the dsRNA-dependent kinase PKR, but seems to require receptor interacting protein 1 as well as Tank-binding kinase 1-mediated activation of the transcription factor IRF-3. The requirement for FADD in mammalian host defence is evocative of innate immune signalling in Drosophila, in which a FADD-dependent pathway responds to bacterial infection by activating the transcription of antimicrobial genes. These data therefore suggest the existence of a conserved pathogen recognition pathway in mammalian cells that is essential for the optimal induction of type I interferons and other genes important for host defence.

OBJECTIVE

Atherosclerosis is an inflammatory disease in which T helper 1 (Th1) immunity has been proposed to play an important role. Nai;ve CD4+ T cells differentiate into interferon-gamma (IFN-gamma) producing Th1 effector cells when stimulated by interleukin-18 (IL-18) and IL-12. We wanted to directly test whether the Th1 pathway is proatherogenic.

METHODS

We bred IL-18(-/-) mice with apolipoprotein E(-/-) (apoE(-/-)) mice and assessed atherosclerosis in the aortic root of the offspring.

RESULTS

24-week-old IL-18 deficient apoE(-/-) mice exhibited substantially reduced lesion size (93,866+/-11273 vs. 144,019+/-9667 microm(2) in IL-18(+/+)xapoE(-/-) mice, P=0.005). Lesion cells in compound knockout mice displayed reduced I-A(b) expression, implying reduced local IFN-gamma stimulation. These mice also had an increased proportion of alpha-SM-actin+ smooth muscle cells, compatible with a more stable lesion phenotype. Immunoglobulin G (IgG) subclass analysis of antibodies to malondialdehyde-modified low density lipoprotein indicated increased Th2 and reduced Th1 helper to B cell antibody production. Surprisingly, serum cholesterol and triglyceride levels were significantly higher in IL-18(-/-)xapoE(-/-) mice in spite of their reduced atherosclerosis. However, no changes in lipoprotein cholesterol patterns were registered.

CONCLUSIONS

These data show reduced atherosclerosis and Th1 activity in spite of increased serum cholesterol in IL-18 deficient apoE(-/-) mice. They support a proatherogenic role for IL-18.

To assess changes in epidermis-derived cytokine mRNA levels early in the afferent phase of allergic contact sensitivity, total epidermal mRNA was analyzed at various times after painting skin with haptens. We used a sensitive reverse transcriptase-polymerase chain reaction technique to quantitatively compare the regulation patterns of the following mRNAs: class II major histocompatibility complex I-A alpha, tumor necrosis factor alpha (TNF-alpha), interleukin (IL) 1 alpha, IL-1 beta, interferon (IFN) gamma, granulocyte/macrophage colony-stimulating factor, IFN-induced protein 10, and macrophage inflammatory protein 2. Enhanced Langerhans cell-derived IL-1 beta mRNA signals were detected as early as 15 min after skin painting with allergens. TNF-alpha, IFN-gamma, and granulocyte/macrophage colony-stimulating factor mRNAs were found to be upregulated after application of allergens, irritant, and tolerogens, but class II major histocompatibility complex I-A alpha, IL-1 alpha, IL-1 beta, IFN-induced protein 10, and macrophage inflammatory protein 2 mRNAs were upregulated only after allergen painting. Depletion of specific cell populations demonstrated that Langerhans cells were the primary source of the IL-1 beta and class II major histocompatibility complex I-A alpha mRNAs, keratinocytes were the primary source of TNF-alpha, IL-1 alpha, IFN-induced protein 10, and macrophage inflammatory protein 2, and infiltrating T lymphocytes were the source of IFN-gamma. Relevance of the molecular findings was demonstrated by the identification of biologically active IL-1 alpha and immunoreactive TNF-alpha in culture supernatants. These studies demonstrate that Langerhans cell-derived and certain keratinocyte-derived cytokine mRNAs are selectively upregulated by allergens in the very early afferent phase of contact sensitivity.

Type I interferons (IFN-alpha/beta) affect many aspects of immune responses. Many pathogen-associated molecules, including bacterial lipopolysaccharide (LPS) and virus-associated double-stranded RNA, induce IFN gene expression through activation of distinct Toll-like receptors (TLRs). Although much has been studied about the activation of the transcription factor IRF-3 and induction of IFN-beta gene by the LPS-mediated TLR4 signaling, definitive evidence is missing about the actual role of IRF-3 in LPS responses in vitro and in vivo. Using IRF-3 deficient mice, we show here that IRF-3 is indeed essential for the LPS-mediated IFN-beta gene induction. Loss of IRF-3 also affects the expression of profile of other cytokine/chemokine genes. We also provide evidence that the LPS/TLR4 signaling activates IRF-7 to induce IFN-beta, if IRF-7 is induced by IFNs prior to LPS simulation. Finally, the IRF-3-deficient mice show resistance to LPS-induced endotoxin shock. These results place IRF-3 as a molecule central to LPS/TLR4 signaling.

Dengue fever is an emerging arboviral disease for which no vaccine or antiviral treatment exists and that causes thousands of fatalities each year. To develop an in vivo test system for antidengue drugs, AG129 mice, which are deficient for the interferon- alpha / beta and - gamma receptors, were injected with unadapted dengue virus, resulting in a dose-dependent transient viremia lasting several days and peaking on day 3 after infection. Additionally, nonstructural protein 1, increased levels of proinflammatory cytokines, and neutralizing IgM and IgG antibodies were found, and mice had splenomegaly. Oral administration of the antiviral compounds 7-deaza-2'-C-methyl-adenosine, N-nonyl-deoxynojirimycin, or 6-O-butanoyl castanospermine significantly reduced viremia in a dose-dependent manner, even after delayed treatment, leading to a reduction of splenomegaly and proinflammatory cytokine levels. The results validate this dengue viremia mouse model as a suitable system for testing antidengue drugs and indicate that antiviral treatment during the acute phase of dengue fever can reduce the severity of the disease.

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacterium capable of causing either acute lethal sepsis or chronic but eventually fatal disease in infected individuals. However, despite the clinical importance of this infection in areas where it is endemic, there is essentially no information on the mechanisms of protective immunity to the bacterium. We describe here a murine model of either acute or chronic infection with B. pseudomallei in Taylor Outbred (TO) mice which mimics many features of the human pathology. Intraperitoneal infection of TO mice at doses of >10(6) CFU resulted in acute septic shock and death within 2 days. In contrast, at lower doses mice were able to clear the inoculum from the liver and spleen over a 3- to 4-week period, but persistence of the organism at other sites resulted in a chronic infection of between 2 and 16 months duration which was eventually lethal in all of the animals tested. Resistance to acute infection with B. pseudomallei was absolutely dependent upon the production of gamma interferon (IFN-gamma) in vivo. Administration of neutralizing monoclonal antibody against IFN-gamma lowered the 50% lethal dose from >5 x 10(5) to ca. 2 CFU and was associated with 8,500- and 4,400-fold increases in the bacterial burdens in the liver and spleen, respectively, together with extensive destruction of lymphoid architecture in the latter organ within 48 h. Neutralization of either tumor necrosis factor alpha or interleukin-12 but not granulocyte-macrophage colony-stimulating factor, also increased susceptibility to infection in vivo. Together, these results provide the first evidence of a host protective mechanism against B. pseudomallei. The rapid production of IFN-gamma within the first day of infection determines whether the infection proceeds to an acute lethal outcome or becomes chronic.

Poliovirus selectively replicates in neurons in the spinal cord and brainstem, although poliovirus receptor (PVR) expression is observed in both the target and nontarget tissues in humans and transgenic mice expressing human PVR (PVR-transgenic mice). We assessed the role of alpha/beta interferon (IFN) in determining tissue tropism by comparing the pathogenesis of the virulent Mahoney strain in PVR-transgenic mice and PVR-transgenic mice deficient in the alpha/beta IFN receptor gene (PVR-transgenic/Ifnar knockout mice). PVR-transgenic/Ifnar knockout mice showed increased susceptibility to poliovirus. After intravenous inoculation, severe lesions positive for the poliovirus antigen were detected in the liver, spleen, and pancreas in addition to the central nervous system. These results suggest that the alpha/beta IFN system plays an important role in determining tissue tropism by protecting nontarget tissues that are potentially susceptible to infection. We subsequently examined the expression of IFN and IFN-stimulated genes (ISGs) in the PVR-transgenic mice. In the nontarget tissues, ISGs were expressed even in the noninfected state, and the expression level increased soon after poliovirus infection. On the contrary, in the target tissues, ISG expression was low in the noninfected state and sufficient response after poliovirus infection was not observed. The results suggest that the unequal IFN response is one of the important determinants for the differential susceptibility of tissues to poliovirus. We consider that poliovirus replication was observed in the nontarget tissues of PVR-transgenic/Ifnar knockout mice because the IFN response was null in all tissues.

Interleukin-1 (IL-1), fibroblast growth factors (FGFs), and their homologues are secreted factors that share a common beta-barrel structure and act on target cells by binding to cell surface receptors with immunoglobulin-like folds in their extracellular domain. While numerous members of the FGF family have been discovered, the IL-1 family has remained small and outnumbered by IL-1 receptor homologues. From expressed sequence tag data base searches, we have now identified four additional IL-1 homologues, IL-1H1, IL-1H2, IL-1H3, and IL-1H4. Like most other IL-1/FGFs, these proteins do not contain a hydrophobic leader sequence. IL-1H4 has a propeptide sequence, while IL-1H1, IL-1H2, and IL-1H3 encode only the mature protein. Circular dichroism spectra and thermal stability analysis suggest that IL-1H1 folds similarly to IL-1ra. The novel homologues are not widely expressed in mammals. IL-1H1 is constitutively expressed only in placenta and the squamous epithelium of the esophagus. However, IL-1H1 could be induced in vitro in keratinocytes by interferon-gamma and tumor necrosis factor-alpha and in vivo via a contact hypersensitivity reaction or herpes simplex virus infection. This suggests that IL-1H1 may be involved in pathogenesis of immune mediated disease processes. The addition of four novel IL-1 homologues suggests that the IL-1 family is significantly larger than previously thought.