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1.
J Immunol ; 174(2): 1003-12, 2005 Jan 15.
Article in English | MEDLINE | ID: mdl-15634924

ABSTRACT

IFN-alphabeta functions in the transition from innate to adaptive immunity and may impinge on the interaction of Mycobacterium tuberculosis with its host. Infection by M. tuberculosis causes IFN-alphabeta secretion and down-regulation of IFN-alphabeta signaling in human APC and the human monocytic cell line THP-1, which provides a model for these studies. Neutralization of secreted IFN-alphabeta prevents inhibition of IFN-alpha signaling during infection, but several lines of evidence distinguish inhibition due to infection from a negative feedback response to only IFN-alphabeta. First, greater inhibition of IFN-alpha-stimulated STAT-1 tyrosine phosphorylation occurs 3 days postinfection than 1 or 3 days after IFN-alphabeta pretreatment. Second, LPS also induces IFN-alphabeta secretion and causes IFN-alphabeta-dependent down-regulation of IFN-alpha signaling, yet the inhibition differs from that caused by infection. Third, IFN-alpha signaling is inhibited when cells are grown in conditioned medium collected from infected cells 1 day postinfection, but not if it is collected 3 days postinfection. Because IFN-alphabeta is stable, the results with conditioned medium suggest the involvement of an additional, labile substance during infection. Further characterizing signaling for effects of infection, we found that cell surface IFN-alphabeta receptor is not reduced by infection, but that infection increases association of protein tyrosine phosphatase 1c with the receptor and with tyrosine kinase 2. Concomitantly, IFN-alpha stimulation of tyrosine kinase 2 tyrosine phosphorylation and kinase activity decreases in infected cells. Moreover, infection reduces the abundance of JAK-1 and tyrosine-phosphorylated JAK-1. Thus, the distinctive down-regulation of IFN-alpha signaling by M. tuberculosis occurs together with a previously undescribed combination of inhibitory intracellular events.


Subject(s)
Down-Regulation/immunology , Feedback , Interferon-alpha/metabolism , Interferon-beta/metabolism , Mycobacterium tuberculosis/immunology , Signal Transduction/immunology , Cell Line, Tumor , Cell Membrane/immunology , Cell Membrane/metabolism , Cell Membrane/microbiology , Culture Media, Conditioned/pharmacology , Cytoplasm/immunology , Cytoplasm/metabolism , Cytoplasm/microbiology , Humans , Interferon-alpha/antagonists & inhibitors , Interferon-alpha/physiology , Interferon-beta/physiology , Intracellular Signaling Peptides and Proteins , Janus Kinase 1 , Lipopolysaccharides/pharmacology , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Protein Tyrosine Phosphatases/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Receptor, Interferon alpha-beta , Receptors, Interferon/metabolism , TYK2 Kinase , Tyrosine/antagonists & inhibitors , Tyrosine/metabolism
2.
Infect Immun ; 72(3): 1275-83, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14977928

ABSTRACT

Gamma interferon (IFN-gamma) is critical in the immune response against Mycobacterium tuberculosis. In an ongoing trial of aerosol IFN-gamma in conjunction with standard drug therapy, we have observed activation of IFN signaling in bronchoalveolar lavage (BAL) cells from tuberculosis (TB) patients. We hypothesized that aerosol IFN-gamma treatment of pulmonary TB would increase expression of genes important for the control of TB. We investigated the expression of downstream genes by measuring inducible nitric oxide synthase (iNOS) and the chemokine IFN-inducible 10-kDa protein (IP-10) by real-time quantitative reverse transcription-PCR. In vitro, M. tuberculosis induced IP-10, and IFN-gamma stimulated this further, with no effect on iNOS expression. We studied 21 patients with pulmonary TB and 7 healthy subjects. Similar to the in vitro model, IP-10 mRNA was increased in BAL cells from TB patients and was augmented after treatment with aerosolized IFN-gamma. TB was also associated with elevated iNOS mRNA, but aerosolized IFN-gamma did not further enhance expression. Genomic analysis identified 1,300 of 4,058 genes expressed in BAL cells from six TB patients before and after 1 month of therapy, including aerosolized IFN-gamma. However, only 15 genes were differentially regulated by IFN-gamma. We conclude that iNOS and IP-10 mRNA expression is increased in TB but that aerosol IFN-gamma treatment increases expression of few genes in the human lung.


Subject(s)
Chemokines, CXC/genetics , Interferon-gamma/pharmacology , Nitric Oxide Synthase/genetics , Tuberculosis, Pulmonary/therapy , Adult , Aerosols , Base Sequence , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Case-Control Studies , Chemokine CXCL10 , DNA, Complementary/genetics , Female , Gene Expression/drug effects , Humans , In Vitro Techniques , Interferon-gamma/administration & dosage , Lung/drug effects , Lung/immunology , Male , Middle Aged , Nitric Oxide Synthase Type II , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins , Tuberculosis, Pulmonary/genetics , Tuberculosis, Pulmonary/immunology
3.
J Immunol ; 172(5): 2935-43, 2004 Mar 01.
Article in English | MEDLINE | ID: mdl-14978096

ABSTRACT

Host defense against Mycobacterium tuberculosis requires the cytokine IFN-gamma and IFN regulatory factor 1 (IRF-1), a transcription factor that is induced to high levels by IFN-gamma. Therefore, we chose to study regulation of IRF-1 expression as a model for effects of M. tuberculosis on response to IFN-gamma. We found that IRF-1 mRNA abundance increased far more than transcription rate in human monocytic THP-1 cells stimulated by IFN-gamma, but less than transcription rate in cells infected by M. tuberculosis. IFN-gamma stimulation of infected cells caused a synergistic increase in IRF-1 transcription, yet IRF-1 mRNA abundance was similar in uninfected and infected cells stimulated by IFN-gamma, as was the IRF-1 protein level. Comparable infection by Mycobacterium bovis bacillus Calmette-Guérin failed to induce IRF-1 expression and had no effect on the response to IFN-gamma. We also examined the kinetics of transcription, the mRNA t(1/2), and the distribution of IRF-1 transcripts among total nuclear RNA, poly(A) nuclear RNA, and poly(A) cytoplasmic RNA pools in cells that were infected by M. tuberculosis and/or stimulated by IFN-gamma. Our data suggest that infection by M. tuberculosis inhibits RNA export from the nucleus. Moreover, the results indicate that regulated entry of nascent transcripts into the pool of total nuclear RNA affects IRF-1 expression and that this process is stimulated by IFN-gamma and inhibited by M. tuberculosis. The ability of infection by M. tuberculosis to limit the increase in IRF-1 mRNA expression that typically follows transcriptional synergism may contribute to the pathogenicity of M. tuberculosis.


Subject(s)
Adjuvants, Immunologic/physiology , DNA-Binding Proteins/genetics , Gene Silencing/immunology , Interferon-gamma/physiology , Mycobacterium tuberculosis/immunology , Phosphoproteins/genetics , RNA Processing, Post-Transcriptional/immunology , Up-Regulation/immunology , Active Transport, Cell Nucleus/genetics , Active Transport, Cell Nucleus/immunology , Adjuvants, Immunologic/antagonists & inhibitors , Cell Line, Tumor , Cell Nucleus/genetics , Cell Nucleus/metabolism , DNA/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/metabolism , Humans , Interferon Regulatory Factor-1 , Interferon-gamma/antagonists & inhibitors , Macrophage Activation/genetics , Macrophage Activation/immunology , Monocytes/immunology , Monocytes/metabolism , Monocytes/microbiology , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/biosynthesis , Phosphoproteins/metabolism , Protein Binding/genetics , Protein Binding/immunology , RNA Stability/immunology , RNA, Messenger/biosynthesis , Transcription, Genetic/immunology , Up-Regulation/genetics
4.
Oncogene ; 22(57): 9121-30, 2003 Dec 11.
Article in English | MEDLINE | ID: mdl-14668793

ABSTRACT

Arsenic trioxide (As2O3) can induce clinical remission in patients with acute promyelocytic leukemia (APL), including those who have relapsed after treatment with all-trans-retinoic acid (RA). In vitro studies with the APL-derived NB4 cell line showed that As2O3 exerts a dose-dependent dual effect, which induces apoptosis at 1 microM, whereas at a lower concentration of 0.1 microM, a partial differentiation of APL is observed. In non-APL cells, interferon (IFN) alpha and 1 microM As2O3 act synergistically to induce apoptosis. In this report, we show that in NB4 cells and in two RA-resistant NB4-derived cell lines, NB4-R1 and NB4-R2, IFNalpha or IFNgamma combined with 0.1 microM As2O3 lead to an increased maturation effect. Moreover, IFNgamma alone is able to differentiate RA-sensitive and -resistant cells with a higher maturation effect on NB4-R2 cells. In contrast, all these cells underwent apoptosis in the presence of the cytokine and a higher concentration of As2O3. IFNgamma boosted As2O3-induced apoptosis in APL cells as tested by TUNEL, Annexin V staining and activation of caspase 3. As2O3 differently altered IFN-induced gene products; it downregulated PML/RARalpha and PML, did not alter PKR and Stat1, and upregulated interferon regulatory family (IRF)-1. Synergism by IFNgamma and arsenic on IRF-1 expression is mediated by a composite element in the IRF-1 promoter that includes an IFNgamma-activation site (GAS) overlapped by a nonconsensus site for nuclear factor kappa B (NFkappaB). Arsenic has no effect on NFkappaB, whereas it enhances the activation of Stat1 by IFNgamma in NB4 cells leading to an increase in IRF-1 expression.


Subject(s)
Apoptosis/drug effects , Arsenic/toxicity , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic/immunology , Interferon-gamma/pharmacology , Oxides/toxicity , Phosphoproteins/genetics , Signal Transduction/physiology , Trans-Activators/metabolism , Arsenic Trioxide , Arsenicals , Cell Differentiation/drug effects , Drug Resistance, Neoplasm , Drug Synergism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Interferon Regulatory Factor-1 , Leukemia, Promyelocytic, Acute , Promoter Regions, Genetic , STAT1 Transcription Factor , Signal Transduction/drug effects , Superoxides/metabolism , Tretinoin/toxicity , Tumor Cells, Cultured
5.
Infect Immun ; 71(5): 2487-97, 2003 May.
Article in English | MEDLINE | ID: mdl-12704120

ABSTRACT

We previously reported that infection by Mycobacterium tuberculosis, the causative agent of tuberculosis, leads to secretion of alpha/beta interferon (IFN-alpha/beta). While IFN-alpha/beta ordinarily stimulates formation of signal transducer and stimulator of transcription-1 (STAT-1) homodimers and IFN-stimulated gene factor-3 (ISGF-3), only ISGF-3 is found in infected human monocytes and macrophages. We have now investigated the basis for this unusual profile of transcription factor activation and its consequences for regulation of transcription, as well as the impact of infection on response to IFN-alpha. After infection, IFN-alpha stimulation of STAT-1 homodimers is inhibited in monocytes and macrophages, while stimulation of ISGF-3 increases in monocytes but tends to decline in macrophages. Effects of infection on the abundance of ISGF-3 subunits, STAT-1, STAT-2, and interferon regulatory factor 9, and on tyrosine phosphorylation of STAT-1 and STAT-2 explain the observed changes in DNA-binding activity, which correlate with increased or inhibited transcription of genes regulated by ISGF-3 and STAT-1. Infection by Mycobacterium bovis BCG does not inhibit IFN-alpha-stimulated tyrosine phosphorylation of STAT-1, formation of homodimers, or transcription of genes regulated by STAT-1 homodimers, suggesting that inhibition of the response to IFN-alpha/beta by M. tuberculosis is an aspect of pathogenicity. Thus, this well-known feature of infection by pathogenic viruses may also be a strategy employed by pathogenic bacteria.


Subject(s)
Interferon-alpha/pharmacology , Mycobacterium tuberculosis/pathogenicity , Cell Line , DNA/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Dimerization , Gene Expression/drug effects , Humans , Interferon-Stimulated Gene Factor 3 , Interferon-Stimulated Gene Factor 3, gamma Subunit , Interferon-alpha/biosynthesis , Phosphorylation , STAT1 Transcription Factor , STAT2 Transcription Factor , Trans-Activators/chemistry , Trans-Activators/metabolism , Transcription Factors/metabolism , Tyrosine/metabolism
6.
Infect Immun ; 71(4): 2058-64, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12654826

ABSTRACT

Tuberculosis is the seventh leading cause of morbidity and mortality in the world, with eight million cases per year. Animal and human studies demonstrate an enrichment of CD4 cells at sites of disease, with a more favorable clinical course when there is a Th1 response with the presence of gamma interferon (IFN-gamma). We previously treated patients who had multidrug-resistant tuberculosis with recombinant IFN-gamma (rIFN-gamma) in aerosol form and were able to convert smear-positive cases to smear negative with 12 treatments over 1 month. We hypothesized that rIFN-gamma would induce signal transducer and activator of transcription (STAT) and interferon regulatory factor (IRF) binding activity in alveolar macrophages (AM). AM treated in vitro showed clear upregulation of STAT-1 and IRF-1 by rIFN-gamma. STAT-1 was not activated and IRF-1 was only weakly induced after 1 day of infection by Mycobacterium tuberculosis TN913. In bronchoalveolar lavage (BAL) cells obtained from 10 of 10 tuberculosis patients 10 +/- 2 days post-antituberculosis treatment, there was no detectable STAT-1 or IRF-1 DNA-binding activity. After 4 weeks of treatment with rIFN-gamma aerosol in addition to the antituberculosis drugs, 10 of 10 patients had increased STAT-1, IRF-1, and/or IRF-9 DNA-binding activity in BAL cells from lung segments shown radiographically to be involved and in those shown to be uninvolved. Symptoms and chest radiographs improved, and amounts of macrophage inflammatory cytokines and human immunodeficiency virus type 1 (HIV-1) viral loads (in five of five HIV-1-coinfected patients) declined in the second BAL specimens. rIFN-gamma aerosol induces signal transduction and gene expression in BAL cells and should be evaluated for efficacy in a randomized, controlled clinical trial.


Subject(s)
Gene Expression Regulation/drug effects , Interferon-gamma/pharmacology , Macrophages, Alveolar/drug effects , Signal Transduction/drug effects , Tuberculosis, Pulmonary/drug therapy , AIDS-Related Opportunistic Infections/drug therapy , AIDS-Related Opportunistic Infections/microbiology , Adult , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , HIV Infections/complications , Humans , Interferon Regulatory Factor-1 , Interferon-Stimulated Gene Factor 3 , Interferon-Stimulated Gene Factor 3, gamma Subunit , Interferon-gamma/administration & dosage , Interferon-gamma/therapeutic use , Macrophages, Alveolar/immunology , Macrophages, Alveolar/microbiology , Male , Middle Aged , Mycobacterium tuberculosis , Phosphoproteins/genetics , Phosphoproteins/metabolism , Recombinant Proteins , STAT1 Transcription Factor , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Treatment Outcome , Tuberculosis, Pulmonary/immunology , Tuberculosis, Pulmonary/microbiology
7.
J Biol Chem ; 277(25): 22377-85, 2002 Jun 21.
Article in English | MEDLINE | ID: mdl-11948194

ABSTRACT

Alveolar macrophages and newly recruited monocytes are targets of infection by Mycobacterium tuberculosis. Therefore, we examined the expression of interferon regulatory factor 1 (IRF-1), which plays an important role in host defense against M. tuberculosis, in undifferentiated and differentiated cells. Infection induced IRF-1 in both. IRF-1 from undifferentiated, uninfected monocytic cell lines was modified during extraction to produce specific species that were apparently smaller than intact IRF-1. After infection by M. tuberculosis or differentiation, intact IRF-1 was recovered. Subcellular fractions were assayed for the ability to modify IRF-1 or inhibit its modification. A serine protease on the cytoplasmic surface of an organelle or vesicle in the "lysosomal/mitochondrial" fraction from undifferentiated cells was responsible for the modification of IRF-1. Thus, the simplest explanation of the modification is cleavage of IRF-1 by the serine protease. Recovery of intact IRF-1 correlated with induction of a serine protease inhibitor that was able to significantly reduce the modification of IRF-1. The inhibitor was present in the cytoplasm of M. tuberculosis-infected or -differentiated cells. It is likely that induction of both IRF-1 and the serine protease inhibitor in response to infection by M. tuberculosis represent host defense mechanisms.


Subject(s)
DNA-Binding Proteins/biosynthesis , Monocytes/microbiology , Mycobacterium tuberculosis/pathogenicity , Phosphoproteins/biosynthesis , Serine Proteinase Inhibitors/pharmacology , Apoptosis , Cell Differentiation , Cell Line , Cells, Cultured , Culture Media, Conditioned/pharmacology , Cytoplasm/metabolism , DNA/metabolism , Enzyme Inhibitors/pharmacology , Humans , Immunoblotting , Interferon Regulatory Factor-1 , Kinetics , Lysosomes/metabolism , Macrophages/metabolism , Mitochondria/metabolism , Serine Endopeptidases/metabolism , Subcellular Fractions
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