IP-10 is a member of the chemokine family of cytokines and is induced in a variety of cells in response to interferon gamma and lipopolysaccharide. The self-aggregation common to many chemokines, including IP-10, has hindered the identification of a specific IP-10 receptor. Using an IP-10 alkaline phosphatase fusion protein that fortuitously blocks this self-aggregation, we have identified an IP-10 binding site on a variety of cells including endothelial, epithelial, and hematopoietic cells. This binding site has a Kd of 25 nM, is inhibited by recombinant murine or human IP-10, and is dependent on the presence of cell surface heparan sulfate proteoglycans (HSPG). This conclusion is based on the findings that IP-10 binding to cells is: (a) inhibited by heparin and heparan sulfate; (b) sensitive to a 1 M NaCl wash; (c) eliminated by treatment with heparinase and trypsin; and (d) absent on mutant CHO cells that do not express cell surface HSPG. Platelet factor 4 (PF4), but not IL-8, monocyte chemoattractant protein-1, RANTES, monocyte inflammatory protein (MIP)-1 alpha, or MIP-1 beta, can compete effectively with IP-10 for binding to the cell surface. Furthermore, IP-10 shares with PF4 the ability to inhibit endothelial cell proliferation (IC50 = 150 nM). These studies demonstrate specificity in the interaction of chemokines and HSPG, and they define IP-10 and PF4 as a distinct subset of chemokines sharing an HSPG-binding site and angiostatic properties.

The beta 2-microglobulin (beta 2m) protein associates with the products of the class I major histocompatibility (MHC) loci; this combination functions in the thymic development of and antigen presentation to CD8+ T cells. Mice in which the beta 2m gene has been disrupted by homologous recombination fail to express class I MHC gene products, and therefore lack CD8+ T cells and measurable cytotoxic T-cell responses. However, beta 2m- mice appear to have normal development of both CD4+ alpha/beta T-cell receptor (TCR+) and gamma/delta TCR+ T cells and are not overtly more susceptible than beta 2m+ mice to potential environmental agents of infection or to experimental viral infection. Here we show that beta 2m- mice suffer high parasitaemias and early death when infected with the obligate cytoplasmic protozoan parasite Trypanosoma cruzi. Despite this increased susceptibility, the beta 2m- mice are more responsive than their beta 2m+ littermates in terms of lymphokine production, making higher levels of both interleukin-2 and interferon-gamma in response to mitogen stimulation. In addition, the beta 2m- mice show essentially no inflammatory response in parasite-infected tissues. These results confirm previous experiments on mice depleted of CD8+ cells using antibody treatment in demonstrating the importance of CD8+ T cells in immune protection in T. cruzi infection. They also implicate CD8+ T cells and/or class I MHC molecules in regulation of lymphokine production and recruitment of inflammatory cells.

Previous studies have shown the implication of beta-defensins in host defense of the human body. The human beta-defensins 1 and 2 (hBD-1, hBD-2) have been isolated by biochemical methods. Here we report the identification of a third human beta-defensin, called human beta-defensin 3 (hBD-3; cDNA sequence, Genbank accession no. AF295370), based on bioinformatics and functional genomic analysis. Expression of hBD-3 is detected throughout epithelia of many organs and in non-epithelial tissues. In contrast to hBD-2, which is upregulated by microorganisms or tumor necrosis factor-alpha (TNF-alpha), hBD-3 expression is increased particularly after stimulation by interferon-gamma. Synthetic hBD-3 exhibits a strong antimicrobial activity against gram-negative and gram-positive bacteria and fungi, including Burkholderia cepacia. In addition, hBD-3 activates monocytes and elicits ion channel activity in biomembranes, specifically in oocytes of Xenopus laevis. This paper also shows that screening of genomic sequences is a valuable tool with which to identify novel regulatory peptides. Human beta-defensins represent a family of antimicrobial peptides differentially expressed in most tissues, regulated by specific mechanisms, and exerting physiological functions not only related to direct host defense.

Epstein-Barr virus (EBV) latency III infection converts B lymphocytes into lymphoblastoid cell lines (LCLs) by expressing EBV nuclear and membrane proteins, EBNAs, and latent membrane proteins (LMPs), which regulate transcription through Notch and tumor necrosis factor receptor pathways. The role of NF-kappa B in LMP1 and overall EBV latency III transcriptional effects was investigated by treating LCLs with BAY11-7082 (BAY11). BAY11 rapidly and irreversibly inhibited NF-kappa B, decreased mitochondrial membrane potential, induced apoptosis, and altered LCL gene expression. BAY11 effects were similar to those of an NF-kappa B inhibitor, Delta N-I kappa B alpha, in effecting decreased JNK1 expression and in microarray analyses. More than 80% of array elements that decreased with Delta N-I kappa B alpha expression decreased with BAY11 treatment. Newly identified NF-kappa B-induced, LMP1-induced, and EBV-induced genes included pleckstrin, Jun-B, c-FLIP, CIP4, and I kappa B epsilon. Of 776 significantly changed array elements, 134 were fourfold upregulated in EBV latency III, and 74 were fourfold upregulated with LMP1 expression alone, whereas only 28 were more than fourfold downregulated by EBV latency III. EBV latency III-regulated gene products mediate cell migration (EBI2, CCR7, RGS1, RANTES, MIP1 alpha, MIP1 beta, CXCR5, and RGS13), antigen presentation (major histocompatibility complex proteins and JAW1), mitogen-activated protein kinase pathway (DUSP5 and p62Dok), and interferon (IFN) signaling (IFN-gamma R alpha, IRF-4, and STAT1). Comparison of EBV latency III LCL gene expression to immunoglobulin M (IgM)-stimulated B cells, germinal-center B cells, and germinal-center-derived lymphomas clustered LCLs with IgM-stimulated B cells separately from germinal-center cells or germinal-center lymphoma cells. Expression of IRF-2, AIM1, ASK1, SNF2L2, and components of IFN signaling pathways further distinguished EBV latency III-infected B cells from IgM-stimulated or germinal-center B cells.

Activation of the inflammatory immune system provokes numerous neuroendocrine and neurotransmitter changes, many of which are similar to those provoked by physical or psychological stressors. These findings, among others, have led to the suggestion that the brain translates immune activation much as if it were a stressor. In this review, I provide synopses of the effects of traditional stressors on the release of corticotropin-releasing hormones at hypothalamic and extrahypothalamic sites, variations of serotonin and its receptors and changes of brain-derived neurotrophic factor (BDNF). These effects are similar to those elicited by activation of the inflammatory immune system, particularly the impact of the immune-signalling molecules interleukin-1 beta, interleukin-6, tumour necrosis factor-alpha and interferon-alpha on neuroendocrine, neurotransmitter and BDNF function. In addition, it is reported that stressors and cytokines may synergistically influence biological and behavioural processes and that these treatments may have long-term ramifications through the sensitization of processes associated with stress responses. Finally, I present an overview of the depressogenic actions of these cytokines in rodent models and in humans, and I provide provisional suggestions (and caveats) about the mechanisms by which cytokines and stressors might culminate in major depressive disorder.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a proapoptotic member of the TNF family of type II membrane proteins, which constitutes one component of T cell cytotoxicity. In this study, we investigated the expression and function of TRAIL in human peripheral blood T (PBT) cells. Although freshly isolated PBT cells did not express a detectable level of TRAIL on their surface, a remarkable TRAIL expression was rapidly induced on the surface of both CD4(+) and CD8(+) PBT cells upon stimulation with anti-CD3 monoclonal antibody and type I interferons (IFNs). This enhancement of TRAIL expression was a unique feature of type I IFNs (IFN-alpha and IFN-beta), and neither type II IFN (IFN-gamma) nor various other cytokines enhanced TRAIL expression on anti-CD3-stimulated PBT cells. Type I IFNs have been used for clinical treatment of renal cell carcinomas (RCCs), and we found that most RCC cell lines were susceptible to TRAIL-induced apoptosis. Type I IFNs substantially augmented cytotoxic activity of anti-CD3-stimulated PBT cells against RCC cell lines in a TRAIL-dependent manner. These results indicate a unique feature of type I IFNs to regulate TRAIL-mediated T cell cytotoxicity, which may be involved in the antitumor effects of type I IFNs against various tumors.

The pathogenesis of the dementia associated with human immunodeficiency virus (HIV) infection is unclear, but has been postulated to be due to indirect effects of HIV infection including the local production of cytokines. To determine which cytokines are produced in the nervous system and to identify any correlations with dementia, cytokine and HIV messenger RNA expression was analyzed by reverse transcriptase-polymerase chain reaction in the brains from 24 HIV-infected patients with and without dementia and 9 HIV-uninfected control subjects. Levels of tumor necrosis factor-alpha messenger RNA were significantly higher and levels of interleukin (IL)-4 messenger RNA were significantly lower in demented compared to nondemented HIV-infected patients. Demented patients also had lower IL-1 beta levels than did nondemented patients. No significant differences were detected in the amounts of leukemia inhibitory factor, IL-6, transforming growth factor-beta 1 and -beta 2, monokine induced by gamma interferon-2 (MIG-2), or interferon-gamma messenger RNAs. IL-10 and IL-2 messenger RNAs were undetectable in all brains examined. Cytokine messenger RNA levels in nondemented HIV-positive patients were similar to those in HIV-negative control subjects. HIV transcripts were more abundant in subcortical white matter than in the basal ganglia, cortex, or deep white matter. Our findings suggest a possible role for tumor necrosis factor-alpha in the development of neurological dysfunction. Increased levels of tumor necrosis factor-alpha messenger RNA were not associated with increased levels of IL-1 beta messenger RNA, suggesting differential regulation of these monokines in acquired immunodeficiency syndrome dementia.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

We sought to determine whether exosome-like vesicles (ELVs) released from adipose tissue play a role in activation of macrophages and subsequent development of insulin resistance in a mouse model.

METHODS

ELVs released from adipose tissue were purified by sucrose gradient centrifugation and labeled with green fluorescent dye and then intravenously injected into B6 ob/ob mice (obese model) or B6 mice fed a high-fat diet. The effects of injected ELVs on the activation of macrophages were determined through analysis of activation markers by fluorescence-activated cell sorter and induction of inflammatory cytokines using an ELISA. Glucose tolerance and insulin tolerance were also evaluated. Similarly, B6 mice with different gene knockouts including TLR2, TLR4, MyD88, and Toll-interleukin-1 receptor (TIR) domain-containing adaptor protein inducing interferon-beta (TRIF) were also used for testing their responses to the injected ELVs.

RESULTS

ELVs are taken up by peripheral blood monocytes, which then differentiate into activated macrophages with increased secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Injection of obELVs into wild-type C57BL/6 mice results in the development of insulin resistance. When the obELVs were intravenously injected into TLR4 knockout B6 mice, the levels of glucose intolerance and insulin resistance were much lower. RBP4 is enriched in the obELVs. Bone marrow-derived macrophages preincubated with recombinant RBP4 led to attenuation of obELV-mediated induction of IL-6 and TNF-alpha.

CONCLUSIONS

ELVs released by adipose tissue can act as a mode of communication between adipose tissues and macrophages. The obELV-mediated induction of TNF-alpha and IL-6 in macrophages and insulin resistance requires the TLR4/TRIF pathway.

Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN-alpha) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A3B(L) inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3B(S) and A3C had no effect. A3B(L) and A3B(S) were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. Moreover, A3G, A3F, and A3B(L), but not A3B(S), induced extensive G-to-A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A3B(L), A3F, and most markedly A3G, which are expressed in liver and up-regulated by IFN-alpha in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV.

Although considerable evidence implicates the cytokine interferon (IFN)-gamma in atherogenesis, the proximal inducers and the range of sources of its expression remain unknown. This study tested the hypothesis that interleukin (IL)-18 regulates IFN-gamma expression during atherogenesis. Indeed, human atheroma in situ expressed IL-18 and elevated levels of its receptor subunits, IL-18Ralpha/beta, compared with nondiseased arterial tissue. IL-18 occurred predominantly as the mature, 18-kD form and colocalized with mononuclear phagocytes (MPhi), while endothelial cells (ECs), smooth muscle cells (SMCs), and MPhi all expressed IL-18Ralpha/beta. Correspondingly in vitro, only MPhi expressed IL-18, while all three cell types displayed the IL-18Ralpha/beta complex constitutively, exhibiting enhanced expression upon stimulation with LPS, IL-1beta, or tumor necrosis factor (TNF)-alpha. IL-18 signaling evoked effectors involved in atherogenesis, e.g., cytokines (IL-6), chemokines (IL-8), intracellular adhesion molecules (ICAM)-1, and matrix metalloproteinases (MMP-1/-9/-13), demonstrating functionality of the receptor on ECs, SMCs, and MPhi. Finally, IL-18, particularly in combination with IL-12, induced the expression of IFN-gamma in cultured MPhi and, surprisingly, in SMCs (but not in ECs). The expression of functional IL-18 and IL-18 receptor on human atheroma-associated ECs, SMCs, and MPhi, and its unexpected ability to induce IFN-gamma expression in SMCs, suggests a novel paracrine proinflammatory pathway operating during atherogenesis.

Rabies virus (RV) of the Rhabdoviridae family grows in alpha/beta interferon (IFN)-competent cells, suggesting the existence of viral mechanisms preventing IFN gene expression. We here identify the viral phosphoprotein P as the responsible IFN antagonist. The critical involvement of P was first suggested by the observation that an RV expressing an enhanced green fluorescent protein (eGFP)-P fusion protein (SAD eGFP-P) (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004) was eliminated in IFN-competent HEp-2 cell cultures, in contrast to wild-type (wt) RV or an RV replicon lacking the genes for matrix protein and glycoprotein. SAD eGFP-P induced transcription of the IFN-beta gene and expression of the IFN-responsive MxA and STAT-1 genes. Similarly, an RV expressing low levels of P, which was generated by moving the P gene to a promoter-distal gene position (SAD DeltaPLP), lost the ability to prevent IFN induction. The analysis of RV mutants lacking expression of truncated P proteins P2, P3, or P4, which are expressed from internal AUG codons of the wt RV P open reading frame, further showed that full-length P is competent in suppressing IFN-beta gene expression. In contrast to wt RV, the IFN-inducing SAD DeltaPLP caused S386 phosphorylation, dimerization, and transcriptional activity of IFN regulatory factor 3 (IRF-3). Phosphorylation of IRF-3 by TANK-binding kinase-1 expressed from transfected plasmids was abolished in wt RV-infected cells or by cotransfection of P-encoding plasmids. Thus, RV P is necessary and sufficient to prevent a critical IFN response in virus-infected cells by targeting activation of IRF-3 by an upstream kinase.

Memory CD8(+) T cells in the lung airways provide protection from secondary respiratory virus challenge by limiting early viral replication. Here, we demonstrate that although airway-resident memory CD8(+) T cells were poorly cytolytic, memory CD8(+) T cells recruited to the airways early during a recall response showed markedly enhanced cytolytic ability. This enhanced lytic activity did not require cognate antigen stimulation, but rather was dependent on STAT1 transcription factor signaling through the interferon-alpha receptor (Ifnar1), resulting in the antigen-independent expression of granzyme B protein in both murine and human virus-specific T cells. Signaling through Ifnar1 was required for the enhanced lytic activity and control of early viral replication by memory CD8(+) T cells in the lung airways. These findings demonstrate that innate inflammatory signals act directly on memory T cells, enabling them to rapidly destroy infected host cells once they enter infected tissues.

Human plasmacytoid dendritic cells (PDC) are key sentinels alerting both innate and adaptive immune responses through production of huge amounts of alpha/beta interferon (IFN). IFN induction in PDC is triggered by outside-in signal transduction pathways through Toll-like receptor 7 (TLR7) and TLR9 as well as by recognition of cytosolic virus-specific patterns. TLR7 and TLR9 ligands include single-stranded RNA and CpG-rich DNA, respectively, as well as synthetic derivatives thereof which are being evaluated as therapeutic immune modulators promoting Th1 immune responses. Here, we identify the first viruses able to block IFN production by PDC. Both TLR-dependent and -independent IFN responses are abolished in human PDC infected with clinical isolates of respiratory syncytial virus (RSV), RSV strain A2, and measles virus Schwarz, in contrast to RSV strain Long, which we previously identified as a potent IFN inducer in human PDC (Hornung et al., J. Immunol. 173:5935-5943, 2004). Notably, IFN synthesis of PDC activated by the TLR7 and TLR9 agonists resiquimod (R848) and CpG oligodeoxynucleotide 2216 is switched off by subsequent infection by RSV A2 and measles virus. The capacity of RSV and measles virus of human PDC to shut down IFN production should contribute to the characteristic features of these viruses, such as Th2-biased immune pathology, immune suppression, and superinfection.

Alpha/beta interferons (IFN-alpha/beta) are key mediators of the innate immune response against viral infection. The ability of viruses to circumvent IFN-alpha/beta responses plays a crucial role in determining the outcome of infection. In a previous study using subgenomic replicons of the Kunjin subtype of West Nile virus (WNV(KUN)), we demonstrated that the nonstructural protein NS2A is a major inhibitor of IFN-beta promoter-driven transcription and that a single amino acid substitution in NS2A (Ala30 to Pro [A30P]) dramatically reduced its inhibitory effect (W. J. Liu, H. B. Chen, X. J. Wang, H. Huang, and A. A. Khromykh, J. Virol. 78:12225-12235). Here we show that incorporation of the A30P mutation into the WNV(KUN) genome results in a mutant virus which elicits more rapid induction and higher levels of synthesis of IFN-alpha/beta in infected human A549 cells than that detected following wild-type WNV(KUN) infection. Consequently, replication of the WNV(KUN)NS2A/A30P mutant virus in these cells known to be high producers of IFN-alpha/beta was abortive. In contrast, both the mutant and the wild-type WNV(KUN) produced similar-size plaques and replicated with similar efficiency in BHK cells which are known to be deficient in IFN-alpha/beta production. The mutant virus was highly attenuated in neuroinvasiveness and also attenuated in neurovirulence in 3-week-old mice. Surprisingly, the mutant virus was also partially attenuated in IFN-alpha/betagamma receptor knockout mice, suggesting that the A30P mutation may also play a role in more efficient activation of other antiviral pathways in addition to the IFN response. Immunization of wild-type mice with the mutant virus resulted in induction of an antibody response of similar magnitude to that observed in mice immunized with wild-type WNV(KUN) and gave complete protection against challenge with a lethal dose of the highly virulent New York 99 strain of WNV. The results confirm and extend our previous original findings on the role of the flavivirus NS2A protein in inhibition of a host antiviral response and demonstrate that the targeted disabling of a viral mechanism for evading the IFN response can be applied to the development of live attenuated flavivirus vaccine candidates.

Effective antiviral agents are urgently needed to combat the possible return of severe acute respiratory syndrome (SARS). Commercial antiviral agents and pure chemical compounds extracted from traditional Chinese medicinal herbs were screened against 10 clinical isolates of SARS coronavirus by neutralisation tests with confirmation by plaque reduction assays. Interferon-beta-1a, leukocytic interferon-alpha, ribavirin, lopinavir, rimantadine, baicalin and glycyrrhizin showed antiviral activity. The two interferons were only active if the cell lines were pre-incubated with the drugs 16 h before viral inoculation. Results were confirmed by plaque reduction assays. Antiviral activity varied with the use of different cell lines. Checkerboard assays for synergy were performed showing combinations of interferon beta-1a or leukocytic interferon-alpha with ribavirin are synergistic. Since the clinical and toxicity profiles of these agents are well known, they should be considered either singly or in combination for prophylaxis or treatment of SARS in randomised placebo controlled trials in future epidemics.

Recombinant human lymphotoxin (LT) was compared with recombinant human tumor necrosis factor (TNF) for direct actions on cultured human endothelial cells (HEC). At equivalent half-maximal concentrations (based on L929 cytotoxicity units) LT and TNF each caused rapid and transient induction (peak 4 to 6 hr) of an antigen associated with leukocyte adhesion (detected by monoclonal antibody H4/18), a rapid but sustained increased expression (plateau 24 hr) of a lymphocyte adhesion structure (ICAM-1), a gradual (plateau 4 to 6 days) increase in expression of HLA-A,B antigens, and gradual (4 to 6 days) conversion of HEC culture morphology from epithelioid to fibroblastoid, an effect enhanced by immune interferon (IFN-gamma). Induction of H4/18 binding by maximal concentrations of LT or TNF could not be augmented by addition of the other cytokine, and 24 hr pretreatment with LT or TNF produced hyporesponsiveness to both mediators for reinduction. H4/18 binding can be transiently induced by tumor-promoting phorbol esters. Pretreatment with either LT or TNF also fully inhibited induction of H4/18 binding by phorbol ester, whereas phorbol ester pretreatment only variably and partially inhibited reinduction by LT or TNF. These actions of LT on endothelium shared with TNF may serve in vivo to promote lymphocyte and inflammatory leukocyte adhesion and transendothelial migration. Recombinant human interleukin 1 species (IL 1 alpha and IL 1 beta) shared many of the actions of LT and TNF and were indistinguishable from each other. However, IL 1 species could be distinguished from LT/TNF by their relative inability to enhance HLA-A,B expression, by their ability to augment H4/18 binding caused by maximally effective concentrations of LT or TNF, and by their inability to inhibit reinduction of H4/18 binding by LT or TNF. In contrast to the actions of LT or TNF, pretreatment with IL 1 alpha or IL 1 beta only partially inhibited induction of H4/18 binding by phorbol ester, and phorbol ester pretreatment consistently, albeit partially, inhibited induction by IL 1 species. These studies suggest that activated T cells through the secretion of LT can in turn activate the local endothelial lining so as to promote homing and extravasation of inflammatory cells. Furthermore, these LT actions can be augmented or complemented by other locally produced mediators such as IFN-gamma or IL 1.

The expression of class I transplantation antigens encoded in the major histocompatibility complex (H-2 in mouse, HLA in man) can be induced by alpha-, beta- and gamma-interferons. Both transcriptional and post-transcriptional mechanisms have been postulated. Recently, a common sequence has been found in the promoter region of several human genes responsive to IFN-alpha. The promoters of H-2Kb and several other mouse class I genes contain a similar interferon response sequence. We show here, in a transient assay, that the H-2Kb promoter can be induced by all three types of interferon and that the interferon response sequence is necessary for induction to occur. However, the response sequence is active only when associated with a functional enhancer sequence which we have recently identified in the promoter of H-2Kb and other class I genes. The combination of these two sequences can render a heterologous promoter responsive to interferon, irrespective of its orientation relative to the cap site.

Interferons produce a variety of biological effects on cells. They induce resistance to virus proliferation, inhibit cell growth, modify cell structure and differentiation, stimulate some immune functions and inhibit others. However, the different interferon (IFN) species may vary in their mechanism of action and, hence, in their relative efficiency for inducing each of the effect. IFN-gamma (type II) appears to show stronger immunoregulatory and growth inhibitory effects than antiviral effects, but this conclusion has been challenged in other reports. The aim of the present work is to compare the action of IFN-gamma and other (type I) interferons on the induction of (2'-5') oligo(A) synthetase which is probably part of the antiviral response and the induction of the histocompatibility HLA-A,-B,-C antigens. We have shown previously that the induction of both proteins is regulated by interferons at the mRNA level, but show here that IFN-gamma from stimulated human lymphocytes and from monkey cells transfected by cloned human IFN-gamma cDNA induced the HLA-A,-B,-C and beta 2-microglobulin mRNAs or proteins at concentrations over 100 times lower than those needed to induce the (2'-5')oligo(A) synthetase and the antiviral state. This difference was not found with IFN-alpha and -beta (type I).

Patients with systemic lupus erythematosus (SLE) have an increased expression of type I interferon (IFN) regulated genes because of a continuous production of IFN-alpha. The cellular and molecular background to this IFN-alpha production has started to be elucidated during the last years, as well as the consequences for the innate and adaptive immune systems. Plasmacytoid dendritic cells (pDC) activated by immune complexes containing nucleic acids secrete type I IFN in SLE. Type I IFN causes differentiation of monocytes to myeloid-derived dendritic cell (mDC) and activation of autoreactive T and B cells. A new therapeutic option in patients with SLE is, therefore, inhibition of IFN-alpha, and recent data from a phase I clinical trial suggests that administration of neutralizing monoclonal antibodies against anti-IFN-alpha can ameliorate disease activity.

With about 350 million virus carriers, hepatitis B virus (HBV) infection remains a major health problem. HBV is a noncytopathic virus causing persistent infection, but it is still unknown whether host recognition of HBV may activate an innate immune response. We describe that upon infection of primary human liver cells, HBV is recognized by nonparenchymal cells of the liver, mainly by liver macrophages (Kupffer cells), although they are not infected. Within 3 hours, this recognition leads to the activation of nuclear factor kappa B (NF-kappaB) and subsequently to the release of interleukin-6 (IL-6) and other proinflammatory cytokines (IL-8, TNF-alpha, IL-1beta), but does not induce an interferon response. The activation of proinflammatory cytokines, however, is transient, and even inhibits responsiveness toward a subsequent challenge. IL-6 released by Kupffer cells after activation of NF-kappaB controls HBV gene expression and replication in hepatocytes at the level of transcription shortly after infection. Upon binding to its receptor complex, IL-6 activates the mitogen-activated protein kinases exogenous signal-regulated kinase 1/2, and c-jun N-terminal kinase, which inhibit expression of hepatocyte nuclear factor (HNF) 1alpha and HNF 4alpha, two transcription factors essential for HBV gene expression and replication.

CONCLUSIONS

Our results demonstrate recognition of HBV patterns by nonparenchymal liver cells, which results in IL-6-mediated control of HBV infection at the transcriptional level. Thus, IL-6 ensures early control of the virus, limiting activation of the adaptive immune response and preventing death of the HBV-infected hepatocyte. This pattern recognition may be essential for a virus, which infects a new host with only a few virions. Our data also indicate that therapeutic neutralization of IL-6 for treatment of certain diseases may represent a risk if the patient is HBV-infected.

Type 1 diabetes mellitus results from an autoimmune destruction of pancreatic beta-cells. Cytokines, such as interleukin-1 beta and interferon-gamma, are putative mediators of immune-induced beta-cell death and, under in vitro conditions, cause beta-cell apoptosis. We have recently shown that interleukin-1 beta + interferon-gamma modifies the expression of >200 genes in beta-cells. Several of these genes are putative targets for the transcription factor nuclear factor-kappa B (NF-kappa B), and in subsequent experiments we showed that NF-kappa B activation is mostly pro-apoptotic in beta-cells. To identify cytokine-induced and NF-kappa B-regulated genes in primary rat beta-cells, we presently combined two experimental approaches: 1) blocking of NF-kappa B activation in cytokine-exposed beta-cells by a recombinant adenovirus (AdI kappa B((SA)2)) containing an inhibitor of NF-kappa B alpha (I kappa Bac) super-repressor (S32A/S36A) and 2) study of gene expression by microarray analysis. We identified 66 cytokine-modified and NF-kappa B-regulated genes in beta-cells. Cytokine-induced NF-kappa B activation decreased Pdx-1 and increased c-Myc expression. This, together with NF-kappa B-dependent inhibition of Glut-2, pro-hormone convertase-1, and Isl-1 expression, probably contributes to the loss of differentiated beta-cell functions. NF-kappa B also regulates several genes encoding for chemokines and cytokines in beta-cells. The present data suggest that NF-kappa B is a key "switch regulator" of transcription factors and gene networks controlling cytokine-induced beta-cell dysfunction and death.

Cytokines play a crucial role in the modulation of inflammatory response in the gastrointestinal tract. Pro-inflammatory cytokines including tumor necrosis factor-alpha, interferon-gamma, interleukin-1beta?IL-1beta?, and interleukin-12 are essential in mediating the inflammatory response, while anti-inflammatory cytokines including interleukin-10 and transforming growth factor-beta are important in the attenuation or containment of inflammatory process. It is increasingly recognized that cytokines have an important physiological and pathological effect on intestinal tight junction (TJ) barrier. Consistent with their known pro-inflammatory activities, pro-inflammatory cytokines cause a disturbance in intestinal TJ barrier, allowing increased tissue penetration of luminal antigens. Recent studies indicate that the inhibition of cytokine induced increase in intestinal TJ permeability has an important protective effect against intestinal mucosal damage and development of intestinal inflammation. In this review, the effects of various pro-inflammatory and anti-inflammatory cytokines on intestinal TJ barrier and the progress into the mechanisms that mediate the cytokine modulation of intestinal TJ barrier are reviewed.

'Protective autoimmunity' refers to a well-controlled anti-self response that helps the body resist neurodegeneration. The response is mediated by autoimmune T cells, which produce cytokines and growth factors. Using an in vitro assay of hippocampal slices, we show that the cytokines interferon-gamma and (especially) interleukin-4, characteristic of pro-inflammatory and anti-inflammatory T cells, respectively, can make microglia neuroprotective. Aggregated beta-amyloid, like bacterial cell wall-derived lipopolysaccharide, rendered the microglia cytotoxic. Cytotoxicity was correlated with a signal transduction pathway that down-regulates expression of class-II major histocompatibility proteins (MHC-II) through the MHC-II-transactivator and the invariant chain. Protection by interleukin-4 was attributed to down-regulation of tumor necrosis factor-alpha and up-regulation of insulin-like growth factor I. These findings suggest that beneficial or harmful expression of the local immune response in the damaged CNS depends on how microglia interpret the threat, and that a well-regulated T-cell-mediated response enables microglia to alleviate rather than exacerbate stressful situations in the CNS.

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