We recently reported that mice with a T cell-restricted expression of a dominant negative form of transforming growth factor beta receptor type II (dnTGFbetaRII) spontaneously develop autoimmune cholangitis that resembles human primary biliary cirrhosis (PBC), including antimitochondrial antibodies (AMAs) and extensive portal CD4(+) and CD8(+) lymphocytic infiltrates. On the basis of these data, we performed a series of experiments to determine whether the pathology was secondary to direct dnTGFbetaRII disruption of the liver and/or alternatively the appearance of autoreactive T cells. First, using dnTGFbetaRIIRag1(-/-) mice, we noted a normal hepatic and biliary structure. Hence, we performed a rigorous series of adoptive transfer studies, transferring Ly5.1(+) unfractionated spleen cell CD4(+) or CD8(+) T cells from dnTGFbetaRII mice into B6/Rag(-/-) (Ly 5.2) recipients. In unmanipulated dnTGFbetaRII mice, there was a marked increase in CD4(+) and CD8(+) T cell biliary infiltrates with AMA. Indeed, B6/Rag(-/-) recipients of dnTGFbetaRII unfractionated cells develop features of liver disease similar to PBC, suggesting that splenic loss of self-tolerance alone is sufficient to cause disease in this model and therefore that there is no specific abnormality in the biliary targets required for appearance of disease. More importantly, adoptive transfer of CD8(+) but not CD4(+) T cells into B6/Rag(-/-) mice led to liver histopathology remarkably similar to PBC, emphasizing a prominent role for CD8 T cell-mediated pathogenesis. In contrast, B6/Rag(-/-) recipients of CD4(+) T cells from dnTGFbetaRII mice predominantly developed inflammatory bowel disease associated with higher levels of serum interferon gamma and tumor necrosis factor alpha.

CONCLUSIONS

These data suggest that in this model of PBC, autoreactive CD8(+) cells destroy bile ducts.

Lung dendritic cells were identified by immunohistochemistry in lung tissue sections from C57BL/6 mice. Following isolation from the lungs using CD11c magnetic beads, the flow cytometric analysis of I-A(b+) and CD11c(+) cells indicated a mixed population of dendritic cells at different stages of maturation, with most expressing an immature phenotype. When cultured for 7 days with recombinant murine granulocyte-macrophage colony-stimulating factor, 99% of cells were CD11c(+) and had a morphology typical of immature dendritic cells. These cells were negative for CD34, CD14, and CD8 alpha antigens but expressed low levels of the myeloid marker F4/80 and moderate levels of MAC3. All expressed high levels of CD11a (LFA-1), CD11b (Mac1), and CD54 antigens, with low levels of class II major histocompatibility complex. Most cells expressed CD80 but only a small percentage of cells were positive for CD40 and CD86. Both overnight and 7-day cultures of lung dendritic cells were able to phagocytose Mycobacterium tuberculosis, and this was associated with the production of interleukin-12 and stimulation of both naïve and immune T cells to produce gamma interferon.

The development and utilization of a tissue culture system for the analysis of quiescent, nonreplicating herpes simplex virus type 1 (HSV-1) genomes is described. It was demonstrated previously that the HSV-1 Vmw65 mutant in1814, which is impaired for immediate early (IE) transcription, was retained for many days in human fetal lung (HFL) fibroblasts in a quiescent 'latent' state. Molecular analysis of the viral genome was not possible, however, due to residual expression of IE proteins and consequent cytotoxicity at high m.o.i. In the study reported here, IE transcription was reduced further by pretreatment of cells with interferon-alpha (IFN-alpha) and by the use of mutant in1820, a derivative of in1814 in which the Vmw110 promoter was replaced by the Moloney murine leukaemia virus (Momulv) enhancer. The Momulv enhancer was not expressed under IE conditions; thus in1820 was more impaired for replication than in1814 and behaved as if deficient for both Vmw65 and Vmw110. In cells pretreated with IFN-alpha and subsequently infected with in1820 cytotoxicity was overcome, enabling a tissue culture system to be developed in which all cells stably retained at least one quiescent viral genome. To assist the analysis of gene expression, in1820 was further modified by insertion of the Escherichia coli lacZ gene controlled by the human cytomegalovirus enhancer (mutant in1883) or the HSV-1 immediate early Vmw110 promoter (in1884). Expression of beta-galactosidase was not detected after infection of IFN-alpha-pretreated cells with in1883 or in1884 but could be induced in almost all cells containing a viral genome, by superinfection of cultures. In1820-derived viruses were retained for at least 9 days and were not reactivated by subculture of cells. A regular arrangement of nucleosomes, as found in cellular chromatin, was not detected on the viral genome at the thymidine kinase locus. The non-linear genome was a template for reactivation with no requirement for prior conversion to a linear form. A small number of remaining linear genomes resulted from incomplete uncoating of input virus.

The role of interferon-gamma in autoimmune diabetes was assessed by breeding a null mutation of the interferon-gamma receptor alpha chain into the nonobese diabetic mouse strain, as well as into a simplified T cell receptor transgenic model of diabetes. In contrast to a previous report on abrogation of the interferon-gamma gene, mutation of the gene encoding its receptor led to drastic effects on disease in both mouse lines. Nonobese diabetic mice showed a marked inhibition of insulitis-both the kinetics and penetrance-and no signs of diabetes; the transgenic model exhibited near-normal insulitis, but this never evolved into diabetes, either spontaneously or after experimental provocation. This failure could not be explained by perturbations in the ratio of T helper cell phenotypes; rather, it reflected a defect in antigen-presenting cells or in the islet beta cell targets.

The common perception that hemoglobin is involved solely in the transport of oxygen and carbon dioxide has been challenged by recent studies with nitric oxide (NO). These studies have shown that the primordial bacterial flavohemoglobin functions to consume NO enzymatically (to protect from nitrosative stress), whereas mammalian hemoglobin functions to deliver NO (thus maximizing oxygen delivery in the respiratory cycle). Here we report that murine macrophages stimulated to produce NO with lipopolysaccharide and interferon-gamma express the betaminor hemoglobin subunit. Consumption of NO, however, was not increased by cytokines or by hemoglobin expression. These data suggest alternative functions for globins in mammalian cells, and they challenge the prevailing view that the expression of alpha- and beta-globin genes is always balanced and coordinated.

The leader protein of Theiler's virus was previously shown to block the production of alpha/beta interferon by infected cells. Here, we observed that expression of the leader protein in infected cells triggered subcellular redistribution of a nucleus-target green fluorescent protein. It enhanced redistribution of the nuclear polypyrimidine tract-binding protein but had no influence on the localization of the nuclear splicing factor SC-35. The leader protein also interfered with trafficking of the cytoplasmic interferon regulatory factor 3, a factor critical for transcriptional activation of alpha/beta interferon genes.

Human tumors can constitutively express cytokines and growth factors, but the extent of this expression has not been investigated. Using 44 different probes to cytokines, growth factors, and their receptors, we tested 21 melanoma and 5 melanocyte cultures for RNA transcript expression by reverse transcriptase-polymerase chain reaction. With 30 amplification cycles, expression of the cytokines interleukin (IL)-1 beta, IL-6, leukemia inhibitory factor (LIF), IL-7, gro alpha, IL-8 and the p35 chain of IL-12 was detected in more than 60% of melanomas. Concomitant receptors for IL-6 and IL-7 were also detected. IL-1 alpha, IL-5, Rantes, IL-10, interferon (IFN)-beta, tumor-necrosis factor (TNF)-alpha, G-colony-stimulating factor (CSF) and GM-CSF were expressed at lower levels. Melanocytes showed greatly reduced cytokine RNA transcripts, and only gro alpha was consistently detected. No expression of IL-2, IL-3, IL-4, IL-9, the p40 chain of IL-12, IFN-alpha or IFN-gamma RNA transcripts was detected in melanomas or melanocytes. The growth factors expressed by melanomas and, after further signal amplification, by melanocytes were transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), TGF-beta, endothelial-cell growth factor (ECGF), basic-fibroblast growth factor (bFGF), nerve growth factor (NGF) and steel. The receptors EGFR, FGFR, NGFRp70 and c-kit were also expressed by melanomas and melanocytes. These results point to new possible autocrine and paracrine pathways in melanoma biology.

Membrane CD14 is involved in lipopolysaccharide (LPS)-induced monocyte activation; it binds LPS, and antibodies against CD14 block the effects of low-dose LPS. It is unknown how LPS regulates its own receptor CD14 in vitro. Therefore, we investigated the effects of LPS on CD14 mRNA and membrane and soluble CD14 (mCD14 and sCD14, respectively) in human monocytes and macrophages. No changes were observed during the first 3 h of LPS stimulation. After 6 to 15 h, LPS weakly reduced CD14 mRNA and mCD14 and transiently enhanced sCD14 release. A 2-day incubation with LPS caused increases in the levels of CD14 mRNA (2-fold), mCD14 (2-fold), sCD14 (1.5-fold), and LPS-fluorescein isothiocyanate binding (1.5-fold); a 5-h incubation with LPS was sufficient to induce the late effects on mCD14 and sCD14. The maximal effect on mCD14 and sCD14 was reached with>> or = 1 ng of LPS per ml; the proportional distribution of the two sCD14 isoforms was not modified by LPS. Besides rough and smooth LPS, lipid A, heat-killed Escherichia coli, lipoteichoic acid, and Staphylococcus aureus cell wall extract (10 micrograms/ml) caused similar increases of mCD14. The LPS effect was blocked by polymyxin B but not by anti-tumor necrosis factor alpha, anti-interleukin-6, anti-gamma interferon, and anti-LPS-binding protein. LPS-induced tumor necrosis factor alpha production was abolished after a second 4-h challenge. In contrast, the LPS-induced increases CD14 mRNA, mCD14, and sCD14 were stronger and appeared earlier after a second LPS challenge. In conclusion, CD14 is transcriptionally upregulated by LPS and other bacterial cell wall constituents.

Physical disruption of an atheromatous lesion often underlies acute coronary syndromes. Matrix-degrading enzymes, eg, matrix metalloproteinases (MMPs), may cause loss in mechanical integrity of plaque tissue that favors rupture. T lymphocytes accumulate at sites where atheromata rupture, but the mechanisms by which these immune cells may contribute to plaque destabilization are unknown. This study tested the hypothesis that the T-lymphocyte surface molecule CD40 ligand (CD40L), recently localized in atherosclerotic plaques, regulates the expression of MMPs in human vascular smooth muscle cells (SMCs), the most numerous cell type in arteries. We report here that stimulated human T lymphocytes induced the expression of the matrix-degrading enzymes, ie, interstitial collagenase (MMP-1), stromelysin (MMP-3), gelatinase B (MMP-9), and activated gelatinase A (MMP-2), in human vascular SMCs by cell contact via CD40 ligation, as demonstrated by Western blot analysis, zymography, and antibody neutralization. Recombinant human CD40L (rCD40L) induced de novo synthesis of MMP-1, MMP-3, and MMP-9 on vascular SMCs and stimulated the expression of these enzymes to a greater extent than did maximally effective concentrations of tumor necrosis factor-alpha or interleukin-1beta, established agonists of MMP expression. Interferon gamma, another T-lymphocyte- derived cytokine, inhibited the induction of MMPs by rCD40L. Immunohistochemical analysis of human coronary atheromata colocalized MMP-1 and MMP-3 with CD40-positive SMCs. These results demonstrated that CD40 ligand, expressed on T lymphocytes, promoted the expression of matrix-degrading enzymes in vascular SMCs and thus established a new pathway of immune-modulated destabilization in human atheromata.

Behcet's disease (BD) is a systemic disorder of recurrent acute inflammation, characterized by major symptoms of oral aphthous ulcers, uveitis, skin lesions and genital ulcers. Involvement of intestines, vessels, and central nervous system (CNS) sometimes leads to a poor prognosis. Patients with BD are known to distribute along the ancient Silk Road. The incidence is relatively higher from eastern Asia to the Mediterranean area as roughly 1-10 patients in 10,000 people, whereas only 1-2 patients in 1,000,000 people in UK and North America. Although etiology of the disease is still unknown, high prevalence of HLA-B51, increased expression of heat shock protein 60 and Th1 dominant immune responses in the patients are considered important in its pathogenesis. Non-infectious neutrophil activation and infection with Streptococcus sanguis and herpes simplex virus would also be associated. Because BD lacks any pathognomonic symptoms and laboratory findings, the diagnosis relies largely upon the criteria proposed by the International Study Group for Behcet's disease in 1990. In Japan, the diagnosis was also made according to the Japanese criteria revised in 1987. Recently, the Behcet's Disease Research Committee of Japan again revised the Japanese criteria in 2003 to avoid overdiagnosis. The new Japanese criteria are introduced in this review. Differential diagnosis excluding Sweet's disease, pemphigus, erythema nodosum and Crohn's disease is important, and positive laboratory data for pathergy test, prick test for dead Streptococci and HLA-B51 are emphasized to make appropriate diagnosis in these criteria. Pathological findings of the disease-affected site such as erythematous nodosum is also stressed. Treatment for the disease has been chosen according to the clinical symptoms. Non-steroidal anti-inflammatory drugs, immunosuppressants, corticosteroids and colchicine are basically introduced. Recently, effects of interferon-alpha/beta, anti-tumor necrosis factor antibody and thalidomide are encouraging, specifically in treatment for the cases with poor prognosis including eye, intestine, vessel and CNS involvement. Low dose weekly administration of methotraxate looks effective for the cases with CNS involvement. Further studies for elucidation of the etiology, improvement of the diagnostic criteria and development of new therapy are needed to conquer the disease.

Plasmacytoid dendritic cells (PDC) represent a highly specialized immune cell subset that produces large quantities of the anti-viral cytokines type I interferons (IFN-alpha and IFN-beta) upon viral infection. PDC employ a member of the family of toll-like receptors, TLR9, to detect CpG motifs (unmethylated CG dinucleotides in certain base context) present in viral DNA. A certain group of CpG motif-containing oligodeoxynucleotides (CpG ODN), CpG-A, was the first synthetic stimulus available that induced large amounts of interferon-alpha (IFN-alpha) in PDC. However, the mechanism responsible for this activity remained elusive. CpG-A is characterized by a central palindrome and poly(G) at the 5' and 3' end. Here we demonstrate that CpG-A self-assembles to higher order tertiary structures via G-tetrad formation of their poly(G) motifs. Spontaneous G-tetrad formation of CpG-A required the palindrome sequence allowing structure formation in a physiological environment. Once formed, G-tetrad-linked structures were stable even under denaturing conditions. Atomic force microscopy revealed that the tertiary structures formed by CpG-A represent nucleic acid-based nanoparticles in the size range of viruses. Similarly sized preformed polystyrene nanoparticles loaded with a CpG ODN that is otherwise weak at inducing IFN-alpha (CpG-B) gained the potency of CpG-A to induce IFN-alpha. Higher ODN uptake in PDC was not responsible for the higher IFN-alpha-inducing activity of CpG-A or of CpG-B-coated nanoparticles as compared with CpG-B. Based on these results we propose a model in which the spatial configuration of CpG motifs as particle is responsible for the virus-like potency of CpG-A to induce IFN-alpha in PDC.

Both type I interferon (IFN-alpha/beta) and type II IFN (IFN-gamma) exert many functions that are restricted to immune cells. Thus, they play critical roles in innate and adaptive immunity. IFN regulatory factor-4 (IRF-4) and IRF-8 (formerly PU.1 interaction partner [Pip] and IFN consensus sequence binding domain [ICSBP], respectively) are immune cell-specific members of the IRF family that regulate the development of myeloid, lymphoid, and dendritic cells. They form a heterodimeric complex with another immune cell-specific transcription factor PU.1-Spi-1 and regulate transcription of genes in the immune system. This review describes the role of the IRF-8-PU.1 complex in modulating IFN signaling in an immune cell-specific manner. Our studies revealed that some but not all IFN-gamma-inducible genes carry an IFN-gamma activation site (GAS) element that contains a binding site for the IRF- 8-PU.1 complex. The IRF-8-PU.1 complex can take part in GAS-mediated transcription and amplify expression of IFN-gamma-responsive genes initiated by Stat1 in macrophages. Similarly, some but not all IFN-alpha/beta-responsive genes are shown to carry an IFN-stimulated response element (ISRE) that contains an IRF-8-PU.1 binding site. The participation of IRF-8-PU.1 in ISRE-mediated transcription results in the augmentation of IFN-stimulated gene factor 3 (ISGF3)-induced transcription in macrophages. Thus, GAS and ISRE elements, classically defined as universal IFN-alpha/beta and IFN-gamma response sequences, are not the same, and some harbor an embedded motif for IRF- 8-PU.1 binding that functions only in immune cells. Accordingly, the IRF-8-PU.1complex provides secondary IFN signaling pathways unique to the immune system. Collectively, the contribution of IRF-8 and PU.1 to IFN-regulated gene expression may in part account for immune cell-specific functions of IFNs.

The vitamin D receptor (VDR) is a nuclear receptor expressed in a number of different cells of the immune system. This study was performed to determine the effect of VDR deficiency on immune function and inflammation of the gastrointestinal tract in a model of inflammatory bowel disease, namely interleukin-10 (IL-10) knockout mice. IL-10 knockout mice were generated which either could or could not respond to vitamin D (double IL-10/VDR knockout; DKO). The distribution and function of lymphocytes in both the primary and secondary lymphoid organs were compared and determined as a function of the severity of intestinal inflammation. DKO mice had normal thymic development and peripheral T-cell numbers at 3 weeks of age, but a week after intestinal disease was detected the thymus was dysplastic with a reduction in cellularity. The atrophy was coupled with increased apoptosis. The spleen weight of DKO mice increased as a result of the accumulation of red blood cells; however, there was a 50% reduction in the numbers of T and B cells. Conversely, the mesenteric lymph nodes were enlarged and contained increased numbers of lymphocytes. The T cells from DKO mice were of a memory phenotype and were hyporesponsive to T-cell receptor stimulation. Colitis in the DKO mice was associated with local and high expression of IL-2, interferon-gamma, IL-1beta, tumour necrosis factor-alpha and IL-12. The primary and secondary lymphoid organs in DKO mice are profoundly altered as a consequence of the fulminating inflammation in the gastrointestinal tract. VDR expression is required for the T cells and other immune cells to control inflammation in the IL-10 KO mice.

Production of cytokines including gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) is an important early-stage host response following infection with intracellular pathogens. Development of immunity to these pathogens is determined to a large extent by the timing and relative level of expression of the cytokines. Numerous studies have shown that early cytokine responses involving interleukin-12 (IL-12) and IFN-gamma are important for resistance to intracellular pathogens, whereas responses involving IL-4 and IL-10 increase host susceptibility. These often-indistinct early cytokine responses influence the differentiation of naïve CD4(+) T helper cells, which later develop into what have commonly been termed Th1- and Th2-type cells. The characterization of CD4(+) T-helper-cell responses as Th1 or Th2 type is based largely on the cytokine profiles during the specific phase and has been used in recent years to account for the innate resistance and susceptibility of different inbred strains of mice to several intracellular pathogens. Studies investigating cytokine production in terms of CD4(+) T-helper-cell polarization in Burkholderia pseudomallei infection have not been undertaken. In this study, we used semiquantitative reverse transcription-PCR to assess induction of cytokine mRNA in liver and spleen of B. pseudomallei-susceptible BALB/c and relatively resistant C57BL/6 mice following infection with virulent B. pseudomallei. The levels of mRNA for IFN-gamma, TNF-alpha, IL-1beta, IL-6, IL-10, and IL-12 increased in both BALB/c and C57BL/6 mice 24 to 36 h after infection. A comparison of BALB/c and C57BL/6 responses revealed the relative levels of expression of mRNA for several of these cytokines, including IFN-gamma, were greater in BALB/c mice, suggesting a role for endotoxic shock and cytokine-mediated immunopathology in the development of acute melioidosis. Early induction of mRNA for the cytokines classically associated with development of Th1- and Th2-type responses was absent or minimal, and induction levels in both strains of mice were similar. During the specific phase, cytokine mRNA profiles occurred as a combination of Th1- and Th2-type patterns. Collectively, these results demonstrate that cytokine mRNA responses in BALB/c and C57BL/6 mice following infection with virulent B. pseudomallei do not develop as polarized Th1- or Th2-type profiles. Considering the role of TNF-alpha and IFN-gamma in the processes of endotoxic shock, these results also indicate that selected cytokines, while important for resistance to B. pseudomallei infection, are also potential contributors to immunopathology and the development of acute fulminating disease.

The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-alpha/beta) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein-lacking approximately half of its carboxy-terminal end-showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.

The antiviral state induced by alpha/beta interferon (IFN-alpha/beta) is a powerful selective pressure for virus evolution of evasive strategies. The paramyxoviruses simian virus 5 (SV5) and human parainfluenza virus 2 (HPIV2) overcome IFN-alpha/beta responses through the actions of their V proteins, which induce proteasomal degradation of cellular IFN-alpha/beta-activated signal transducers and activators of transcription STAT1 and STAT2. SV5 infection induces STAT1 degradation and IFN-alpha/beta inhibition efficiently in human cells but not in mouse cells, effectively restricting SV5 host range. Here, the cellular basis for this species specificity is demonstrated to result from differences between human and murine STAT2. Expression in mouse cells of full-length or truncated human STAT2 cDNA is sufficient to permit antagonism of endogenous murine IFN-alpha/beta signaling by SV5 and HPIV2 V proteins. Furthermore, virus-induced STAT protein degradation is observed in mouse cells only in the presence of ectopically expressed human STAT2. The results indicate that STAT2 acts as an intracellular determinant of paramyxovirus host range restriction, which contributes to the species specificity of virus replication, and that human STAT2 can confer a growth advantage for SV5 in the murine host.

Type I interferons (IFNs) are potent regulators of normal hematopoiesis in vitro and in vivo, but the mechanisms by which they suppress hematopoietic progenitor cell growth and differentiation are not known. In the present study we provide evidence that IFN alpha and IFN beta induce phosphorylation of the p38 mitogen-activated protein (Map) kinase in CD34+-derived primitive human hematopoietic progenitors. Such type I IFN-inducible phosphorylation of p38 results in activation of the catalytic domain of the kinase and sequential activation of the MAPK-activated protein kinase-2 (MapKapK-2 kinase), indicating the existence of a signaling cascade, activated downstream of p38 in hematopoietic progenitors. Our data indicate that activation of this signaling cascade by the type I IFN receptor is essential for the generation of the suppressive effects of type I IFNs on normal hematopoiesis. This is shown by studies demonstrating that pharmacological inhibitors of p38 reverse the growth inhibitory effects of IFN alpha and IFN beta on myeloid (colony-forming granulocytic-macrophage) and erythroid (burst-forming unit-erythroid) progenitor colony formation. In a similar manner, transforming growth factor beta, which also exhibits inhibitory effects on normal hematopoiesis, activates p38 and MapKapK-2 in human hematopoietic progenitors, whereas pharmacological inhibitors of p38 reverse its suppressive activities on both myeloid and erythroid colony formation. In further studies, we demonstrate that the primary mechanism by which the p38 Map kinase pathway mediates hematopoietic suppression is regulation of cell cycle progression and is unrelated to induction of apoptosis. Altogether, these findings establish that the p38 Map kinase pathway is a common effector for type I IFN and transforming growth factor beta signaling in human hematopoietic progenitors and plays a critical role in the induction of the suppressive effects of these cytokines on normal hematopoiesis.

A partial complementary DNA was isolated for a gene (rrg) that is normally expressed in mouse NIH 3T3 cells, but is down-regulated after cellular transformation by long terminal repeat (LTR)-activated c-H-ras (LTR-c-H-ras). This gene was reexpressed in a nontumorigenic persistent revertant cell line created by prolonged treatment of the transformed cells with mouse interferon alpha/beta. Persistent revertants stably transfected with rrg complementary DNA antisense expression vectors appeared transformed, had decreased amounts of rrg messenger RNA, and were tumorigenic in nude mice. Stable transfection with sense constructs did not alter the normal morphology, message level, or nontumorigenicity of the persistent revertant cell line.

OBJECTIVE

To investigate the production of cytokines by T cells in patients with rheumatoid arthritis (RA), reactive arthritis (REA) and osteoarthritis (OA).

METHODS

The lymphokines interleukin (IL)-2, IL-4, interferon gamma (IFN-gamma) and tumour necrosis factor beta (TNF-beta), as well as the monokines IL-1, IL-6 and TNF-alpha, were measured by immunoassays in sera and synovial fluid (SF) from patients with RA, REA and OA. In addition, cytokine expression was studied by immunohistochemistry in synovial membrane tissue sections from patients with RA and OA.

RESULTS

Almost 60% of RA sera contained at least one of the cytokines investigated, though in low concentrations, whereas cytokines were generally not detectable in sera from REA and OA patients. In contrast, cytokines were found in virtually all SF; thus, the majority of SF from RA patients contained IFN-gamma (median level 17 pg/ml) in addition to the monokines IL-6 (4700 pg/ml) and TNF-alpha (157 pg/ml). IFN-gamma and IL-6 (but not TNF-alpha) were also frequently measured in SF from REA patients, whereas OA samples typically contained only IL-6. Immunohistochemical analysis of tissue sections from RA patients revealed lymphokine expression in 0.1-0.3% of T cells, particularly IL-2 and IFN-gamma, and to a lesser extent also IL-4. Interestingly, the expression of TNF-alpha and IL-6 by synovial T cells was also observed. The majority of cytokine-expressing T cells were CD4-positive T-helper cells typically found in perivascular areas, whereas cytokine-producing CD8-positive T cells were found distributed throughout the synovium. As expected, in specimens from OA patients, T cells were much less abundant and expression of cytokines could not be detected.

CONCLUSIONS

These data clearly demonstrate production of cytokines by T cells in RA synovial tissue, indicating that activated T cells play a role in the pathophysiological events of RA.

In a wide range of human diseases of inflammatory nature like Crohn's disease, pathology is mediated in part by pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF) or interferon-gamma. We show here that a commonly used generic antidepressant bupropion, in wide use worldwide to treat depression in humans for a decade now, profoundly lowers levels of TNF, interferon-gamma, and interleukin-1 beta in vivo, in a mouse lipopolysaccharide (LPS) induced inflammation model. Mice challenged with an otherwise lethal dose of LPS were protected by bupropion and levels of the anti-inflammatory cytokine interleukin-10 were increased. Previous data in rodents and humans indicate antidepressant effects of bupropion are mediated by its weak reuptake inhibition of norepinephrine and dopamine. Concordant with this, TNF suppression by bupropion in our mouse LPS model was largely abrogated by beta-adrenergic or dopamine D1 receptor antagonists but not by a D2 antagonist. TNF synthesis is controlled by an inverse relationship with intracellular cyclic adenosine monophosphate (cAMP) and stimulation of either beta-adrenoreceptors or D1 dopaminergic receptors result in increased cAMP but stimulation of D2 receptors lowers cAMP. We conclude that bupropion may suppress TNF synthesis by mediating increased signaling at beta-adrenoreceptors and D1 receptors, resulting in increased cAMP that inhibits TNF synthesis. Bupropion is well tolerated also in non-psychiatric populations and has less risk with long term use than current anti-inflammatory, immunosuppressive or TNF suppressive treatments such as prednisone, azathioprine, infliximab, or methotrexate. New anti-inflammatory treatments are needed. We believe a new chapter in antiinflammatory, TNF lowering treatment of disease has been opened. Bupropion's use for this in humans should be explored.

OBJECTIVE

To investigate the in vivo acute phase molecular response of the brain to ionizing radiation.

METHODS

C3Hf/Sed/Kam mice were given midbrain or whole-body irradiation. Cerebral expression of interleukins (IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6), interferon (IFN-gamma), tumor necrosis factors (TNF-alpha and TNF-beta), intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthetase (iNOS), von Willebrand factor (vWF), alpha 1-antichymotrypsin (EB22/5.3), and glial fibrillary acidic protein (GFAP) was measured at various times after various radiation doses by ribonuclease (RNase) protection assay. The effects of dexamethasone or pentoxifylline treatment of mice on radiation-induced gene expression were also examined.

RESULTS

Levels of TNF-alpha, IL-1 beta, ICAM-1, EB22/5.3 and to a lesser extent IL-1 alpha and GFAP, messenger RNA were increased in the brain after irradiation, whether the dose was delivered to the whole body or only to the midbrain. Responses were radiation dose dependent, but were not found below 7 Gy; the exception being ICAM-1, which was increased by doses as low as 2 Gy. Most responses were rapid, peaking within 4-8 h, but antichymotrypsin and GFAP responses were delayed and still elevated at 24 h, by which time the others had subsided. Pretreatment of mice with dexamethasone or pentoxifylline suppressed radiation-induced gene expression, either partially or completely. Dexamethasone was more inhibitory than pentoxifylline at the doses chosen.

CONCLUSIONS

The initial response of the brain to irradiation involves expression of inflammatory gene products, which are probably responsible for clinically observed early symptoms of brain radiotherapy. This mechanism explains the beneficial effects of the clinical use of steroids in such circumstances.

Dengue virus (DV) primarily infects blood monocytes (MO) and tissue macrophages (M phi). We have shown in the present study that DV can productively infect primary human MO/M phi regardless of the stage of cell differentiation. After DV infection, the in vitro-differentiated MO/M phi secreted multiple innate cytokines and chemokines, including tumor necrosis factor alpha, alpha interferon (IFN-alpha), interleukin-1 beta (IL-1 beta), IL-8, IL-12, MIP-1 alpha, and RANTES but not IL-6, IL-15, or nitric oxide. Secretion of these mediators was highlighted by distinct magnitude, onset, kinetics, duration, and induction potential. A chemokine-to-cytokine hierarchy was noted in the magnitude and induction potential of secretion, and a chemokine-to-cytokine-to-chemokine/Th1 cytokine cascade could be seen in the production kinetics. Furthermore, we found that terminally differentiated MO/M phi cultured for more than 45 days could support productive DV infection and produce innate cytokines and chemokines, indicating that these mature cells were functionally competent in the context of a viral infection. In addition, DV replication in primary differentiated human MO/M phi was enhanced and prolonged in the presence of lipopolysaccharide (LPS), and LPS-mediated synergistic production of IFN-alpha could be seen in DV-infected MO/M phi. The secretion of innate cytokines and chemokines by differentiated MO/M phi suggests that regional accumulation of these mediators may occur in various tissues to which DV has disseminated and may thus result in local inflammation. The LPS-mediated enhancement of virus replication and synergistic IFN-alpha production suggests that concurrent bacterial infection may modulate cytokine-mediated disease progression during DV infection.

Conjugated linoleic acid (CLA) is a dietary fatty acid that has received considerable attention due to its unique properties in rodent models including anti-cancer, anti-atherogenic and anti-diabetic effects. The effects of CLA are similar to those seen with ligands for peroxisome proliferator-activated receptor (PPARs), most notably of the PPAR gamma subtype. With the recent observation of a role for PPAR gamma in regulation of immune responses, we suspected that CLA could affect immune function, in particular macrophage activity. The goal of our study was to examine whether this dietary fatty acid has anti-inflammatory properties similar to those reported for PPAR gamma activators such as 15-deoxy prostaglandin J(2) (PGJ(2)). In reporter assays, various CLA isomers activated PPAR gamma in RAW264.7 mouse macrophage (RAW) cells. CLA decreased the interferon-gamma (IFN gamma)-induced mRNA expression of mediators of inflammation including cyclooxygenase 2 (COX2), inducible NOS (iNOS), and tumor necrosis factor alpha (TNFalpha). Reporter assays also demonstrated reduced IFN gamma-stimulated transcriptional activity of the iNOS and COX2 promoters by CLA. Consequently, CLA decreased the production of PGE(2), TNFalpha and the inflammatory agent nitric oxide (NO) in RAW cells treated with IFN gamma. Other pro-inflammatory cytokines such as IL-1 beta and IL-6 were similarly decreased by CLA treatment of RAW cells. In addition, various CLA isomers induced HL60 cell differentiation along the monocytic lineage as assessed by measuring expression of the cell surface marker CD14. This differentiation process, as well as the regulation of iNOS and COX2 by 15dPGJ(2), is believed to involve PPAR gamma. Mutations of Leu(468) and Glu(471) to alanine in helix 12 of the ligand-binding domain of PPAR gamma resulted in a protein with strong dominant-negative activity (dnPPAR gamma). Transfecting dnPPAR gamma into RAW cells eliminated the ability of various CLA isomers to regulate the iNOS reporter construct. Taken together, these results suggest that CLA has anti-inflammatory properties that are mediated, at least in part, by the nuclear hormone receptor PPAR gamma.

Interferons (IFNs) are a family of multifunctional cytokines with antiviral activities. The K9 open reading frame of Kaposi's sarcoma-associated herpesvirus (KSHV) exhibits significant homology with cellular IFN regulatory factors (IRFs). We have investigated the functional consequence of K9 expression in IFN-mediated signal transduction. Expression of K9 dramatically repressed transcriptional activation induced by IFN-alpha, -beta, and -gamma. Further, it induced transformation of NIH 3T3 cells, resulting in morphologic changes, focus formation, and growth in reduced-serum conditions. The expression of antisense K9 in KSHV-infected BCBL-1 cells consistently increased IFN-mediated transcriptional activation but drastically decreased the expression of certain KSHV genes. Thus, the K9 gene of KSHV encodes the first virus-encoded IRF (v-IRF) which functions as a repressor for cellular IFN-mediated signal transduction. In addition, v-IRF likely plays an important role in regulating KSHV gene expression. These results suggest that KSHV employs an unique mechanism to antagonize IFN-mediated antiviral activity by harboring a functional v-IRF.