Analysis of transcription factors regulating induction of indoleamine 2,3-dioxygenase by IFN-gamma.

IFN-gamma treatment of the human carcinoma cell line ME180 causes cell death due to induction of indoleamine 2,3-dioxygenase (IDO) and resulting starvation for tryptophan. A mutant cell line 3B6A derived from ME180 was resistant to IFN-gamma because of loss of IDO activity. Cotransfecting an IDO promoter-chloramphenicol acetyl transferase (CAT) construct with IFN regulatory factor-1 (IRF-1) resulted in induction of CAT activity in both ME180 and 3B6A cells even in the absence of IFN-gamma. This induction was reduced by cotransfection with IRF-2. However, IRF-1 was not able to restore IDO activity, suggesting a possible repressor site outside the IDO promoter region. Stat1alpha (p91) restored both CAT and IDO activities in 3B6A cells following IFN-gamma treatment. 3B6A cells doubly treated with IFN-gamma and IFN-alpha or IFN-beta restored IDO activity, although neither cytokine on its own could induce IDO. Western blot analysis showed that both constitutive expression and induction of Stat1alpha by IFN-gamma were reduced in 3B6A cells, and double treatment of IFN-gamma with IFN-alpha or IFN-beta restored the expression level of Statla. Electrophoretic mobility shift assays indicated that Stat1 binds to the IFN-gamma-activated sequence (GAS) region in the IDO promoter in ME180 cells following IFN-gamma treatment. Our results indicated that the defect in 3B6A cells was reduced expression of Stat1alpha and that IRF-1, NF-kappaB, and PKR were all involved to some extent in the induction of IDO following IFN-gamma treatment.     

Specific subsets of murine dendritic cells acquire potent T cell regulatory functions following CTLA4-mediated induction of indoleamine 2,3 dioxygenase

Murine dendritic cells (DCs) expressing indoleamine 2,3 dioxygenase (IDO) catabolize tryptophan and can suppress T cell responses elicited in vivo. Here, we identify specific subsets of splenic (CD11c+) dendritic cells competent to mediate IDO-dependent T cell suppression following CTLA4-mediated ligation of B7 molecules. IDO-competent DC subsets acquired potent and dominant T cell suppressive properties as a consequence of IDO up-regulation, as they blocked the ability of T cells to respond to other stimulatory DCs in the same cultures. Soluble CTLA4 (CTLA4-Ig) and cloned CTLA4+ regulatory T cells (Tr1D1) up-regulated IDO selectively in DC subsets co-expressing B220 or CD8alpha. The ability of Tr1D1 T cells to suppress CD8+ T cell responses was completely dependent on their ability to induce tryptophan catabolism in DCs. Selective IDO up-regulation in DCs did not inhibit T cell activation, but prevented T cell clonal expansion due to rapid death of activated T cells. T cell responses were restored by genetic or pharmacologic inhibition of IDO enzyme activity, or by adding excess tryptophan. DCs from interferon gamma (IFNgamma)-receptor-deficient mice were effective in promoting IDO-dependent T cell suppression following CTLA4-Ig exposure in vivo, indicating that IFNgamma signaling was not necessary for IDO up-regulation in this model. These findings suggest that IDO-competent DCs provide a regulatory bridge, mediated by CTLA4-B7 engagement, between certain regulatory T cell subsets and naive responder T cells.     

The role of IFN-gamma in surgical patients.

Interferon-gamma (IFN-gamma) was shown to play a central role in the regulation of an adequate immune response to trauma in a variety of human in vitro studies and in animal models. However, clinical studies in surgical patients after various injuries failed to clearly prove efficacy despite the fact that all studies have shown enthusiastic in vitro effects. This review summarizes the available data about IFN-gamma after elective operations and accidental trauma and speculates about the further therapeutic potential of this cytokine.     

The role of interferon regulatory factor-1 and interferon regulatory factor-2 in IFN-gamma growth inhibition of human breast carcinoma cell lines.

Interferon (IFN) regulatory factor-1 (IRF-1) and IRF-2 play opposing roles in the regulation of many IFN-gamma-inducible genes. To investigate the signal transduction pathway in response to IFN-gamma in light of differences in growth effects, we selected four human breast carcinoma cell lines based on a spectrum of growth inhibition by IFN-gamma. MDA468 growth was markedly inhibited by IFN-gamma, and it showed substantial induction of IRF-1 mRNA but little IRF-2 induction. SKBR3 showed little growth inhibition and little induction of IRF-1 mRNA but significant induction of IRF-2 mRNA. HS578T and MDA436 growth inhibition and IRF-1/IRF-2 induction were intermediate. All four cell lines showed intact receptor at the cell surface and Stat1 translocation to the nucleus by immunostaining. By EMSA, there were marked differences in the induced ratio of IRF-1 and IRF-2 binding activity between the cell lines that correlated with growth inhibition. Finally, antisense oligonucleotides specific for IRF-1 attenuated IFN-gamma growth inhibition in MDA436 and MDA468, confirming the direct role of IRF-1 in IFN-gamma growth inhibition. Induction of IRF-1 causes growth inhibition in human breast cancer cell lines, and induction of IRF-2 can oppose this. The relative induction of IRF-1 to IRF-2 is a critical control point in IFN-gamma response.     

Tumor necrosis factor-alpha and lipopolysaccharide enhance interferon-induced antichlamydial indoleamine dioxygenase activity independently.

In macrophages, interleukin-1 (IL-1) and lipopolysaccharide (LPS) enhance the antichlamydial effect of interferon-gamma (IFN-gamma) by increasing indoleamine 2,3-dioxygenase (IDO) activity in a dose-dependent manner. Our objectives were to characterize the antichlamydial effect of tumor necrosis factor-alpha (TNF-alpha) on IFN-induced IDO activity and to establish the relationship between LPS and TNF-alpha in IDO potentiation. TNF-alpha inhibited chlamydial growth in a dose-dependent manner only in IFN-treated macrophages. Furthermore, excess tryptophan reversed the effect of combined cytokine treatment, indicating that IDO alone was responsible for chlamydial inhibition. The promonocyte THP-1 cell line, previously used to model the effect of IL-1 on IDO mRNA expression, was treated with IFN-gamma and increasing concentrations of LPS or TNF-alpha. IDO mRNA was quantified by RT-PCR, and IDO activity was measured by HPLC at 24 and 48 h after treatment, respectively. Both LPS and TNF-alpha enhanced IDO activity and IDO mRNA expression, with maximal IDO induction at 100 ng/ml LPS or 5 ng/ml TNF-alpha. Anti-TNF-alpha failed to neutralize the effects of LPS treatment, and insufficient TNF-alpha or IL-1 was produced by LPS-treated THP-1 cells to account for the enhancing effect of LPS, indicating that the effect of LPS on IDO was independent of TNF-alpha and IL-1.     

Antigen-specific regulatory T cells--their induction and role in infection

In addition to the well-established role of natural CD4(+)CD25(+) regulatory T (Tr) cells in the maintenance of tolerance to self-antigens, there is accumulating evidence for distinct populations of Tr cells induced in the periphery after encounter with pathogens and foreign antigens. These antigen-specific T cells, termed Tr1 or Th3 cells, secrete IL-10 and or TGF-beta, but no IL-4 and little or no IFN-gamma, and are induced by semi-mature dendritic cells under the influence of regulatory cytokines, including IL-10, TGF-beta and IL-4. Tr1 or Th3 cells are capable of suppressing Th1 and Th2 responses and function in infection to limit pathogen-induced immunopathology, but can also be exploited in therapies for immune-mediated diseases.     

Effect of 3-hydroxyanthranilic acid in the immunosuppressive molecules indoleamine dioxygenase and HLA-G in macrophages

Indoleamine 2,3-dioxygenase (IDO) and human leukocyte antigen-G (HLA-G) are two molecules involved in immune tolerance. 3-Hydroxyanthranilic acid is an IDO downstream metabolite that can produce an important immune suppression. In dendritic cells, it induces HLA-G cell surface expression and secretion to the medium. The relationship between IDO and HLA-G seems to be dependent on the cell type. In this study we analyzed the effect of the tryptophan metabolite 3-hydroxyanthranilic acid in these two proteins in monocytes and macrophages. This compound decreased IDO activity while increased HLA-G surface expression in macrophages, but not in monocytes. Also, 3-hydroxyanthranilic acid decreased HLA-G1 shedding, but not HLA-G5 secretion by macrophages. These results stress the importance of 3-hydroxyanthranilic acid as a modulator of the immune response.     

IFN-gamma induction of osteopontin expression in human monocytoid cells.

Osteopontin (OPN) is an arginine-glycine-aspartate (RGD)-containing cytokine that increases macrophage expression of the Th1 cytokines interferon-gamma (IFN-gamma) and interleukin-12 (IL-12), and downregulates macrophage expression of IL-10. OPN has been implicated in the clearance of mycobacterial infection and in granuloma formation. We previously showed decreased OPN expression in the granulomas of patients with disseminated mycobacterial infection in the setting of mutation of the IFN-gamma receptor-1 (IFNGR-1). These data suggested that IFN-gamma critically regulates OPN expression during mycobacterial infection. Herein, we characterize the effect of IFN-gamma stimulation on OPN expression by human monocytoid cells. By Western blot, ELISA, and Northern blot analysis, IFN-gamma treatment of THP-1 cells was shown to induce OPN mRNA and protein expression in a time-dependent and dose-dependent manner. The human OPN promoter was amplified by PCR using human genomic DNA as a template and transfected into THP-1 cells. Compared with control, OPN promoter activity increased by 15-fold after treatment with IFN-gamma. Similar induction of OPN was seen in IFN-gamma-stimulated primary human blood monocytes. These data show that IFN-gamma stimulates OPN expression from monocytoid cells and suggest that OPN may function in a positive feedback loop in Th1 inflammation, increasing expression of IFN-gamma, which itself upregulates the OPN gene.     

The role of IFN-gamma in the outcome of chlamydial infection

Chlamydia are intracellular bacteria which infect many vertebrates, including humans. They cause a myriad of severe diseases, ranging from asymptomatic infection to pneumonia, blindness or infertility. IFN-gamma plays an important role in defense against acute infection and in the establishment of persistence. Chlamydia have evolved mechanisms to escape IFN-gamma functions. IFN-gamma-mediated effector mechanisms may involve effects on the metabolism of tryptophan or iron, on the inducible NO synthase (iNOS), on the secretion of chemokines and adhesion molecules or on the regulation of T-cell activities. IFN-gamma is secreted by the innate and the adaptive arms of the immune system. Within the former, Chlamydia-infected macrophages express IFN-gamma that in turn mediates resistance to infection. IFN-alpha/beta are pivotal for both IFN-gamma- and iNOS-mediated resistance to chlamydial infection in macrophages.     

Neem leaf glycoprotein overcomes indoleamine 2,3 dioxygenase mediated tolerance in dendritic cells by attenuating hyperactive regulatory T cells in cervical cancer stage IIIB patients

Tolerogenic dendritic cells (DCs) are a subset of DCs characterized by abundant indoleamine 2,3 dioxygenase (IDO) expressions. IDO may be co-operatively induced in DCs by regulatory T (Tregs) cells and various DC maturation agents. Tregs are markedly amplified in the physiological system of cancer patients, inducing over tolerance in DCs that leads to the hyper accumulation of immunosuppressive IDO in tumor microenvironment, thereby, hampering anti-tumor immunity. Consequently, a major focus of current immunotherapeutic strategies in cancer is to minimize IDO, which is possible by reducing Tregs and using various IDO inhibitors. Neem leaf glycoprotein (NLGP), a natural and nontoxic immunomodulator, demonstrated several unique immunoregulatory activities. Noteworthy activities of NLGP are to mature DCs and to inhibit Tregs. As Tregs are inducer of IDO in DCs and hyperactive Tregs is a hallmark of cancer, we anticipated that NLGP might abrogate IDO induction in DCs by inhibiting Tregs. Evidences are presented here that in a co-culture of DCs and Tregs isolated from cervical cancer stage IIIB (CaCx-IIIB) patients, NLGP does inhibit IDO induction in DCs by curtailing the over expression of Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) on Tregs and concomitantly induces optimal DC maturation. In contrast, in the presence of LPS as maturation agent the DCs displays a tolerogenic profile. This finding suggests the reduction of tolerogenecity of DCs in CaCx-IIIB patients by reducing the IDO pool using NLGP. Accordingly, this study sheds more light on the diverse immunomodulatory repertoire of NLGP.     

Positive regulatory elements involved in urea amidolyase and urea uptake induction in Saccharomyces cerevisiae

Summary Urea amidolyase and the high affinity urea uptake system are induced by allophanate. durM ^– and durL ^– recessive mutations, which are easily obtained, totally prevent this induction. They are not linked to each other nor to the concerned structural genes. Despite an intensive hunt, no mutation of repressor or classical operator type has been selected. We conclude that urea amidolyase and urea uptake induction involves at least two positive elements coded for by the durM and durL genes.     

The role of IFN-gamma in murine Salmonella typhimurium infection

Influence of both recombinant interferon gamma (r-IFN-gamma) and monoclonal anti-IFN-gamma on murine Salmonella typhimurium infection was studied in vivo. As a challenge we used either a virulent or an avirulent strain of S. typhimurium. The avirulent strain is unable to replicate in the mouse spleen. The effect of IFN-gamma or anti-IFN-gamma treatment on infection was assayed by counting the number of bacteria in the spleens 1, 2, 4 or 7 days after infection. Exogenously supplied IFN-gamma was found to decrease the number of both the virulent and the avirulent bacteria in the spleens 1 day after challenge but thereafter had no detectable effect. Anti-IFN-gamma-treated mice were unable to clear a sub-lethal dose of the virulent bacteria. The enhanced growth of the bacteria in the spleen was seen after 2 days of infection. In the spleens of anti-IFN-gamma-treated mice and control mice the fate of the non-replicating bacteria was similar. We conclude that the mice produce IFN-gamma during the early phase of Salmonella infection and that this causes a reduction in the apparent growth rate of the virulent bacteria. Because the avirulent bacteria, which do not multiply, were not affected by anti-IFN-gamma treatment, the effect of IFN-gamma is primarily bacteriostatic rather than bactericidal. After challenge, the production of IFN-gamma seems to start with a lag of approximately 2 days.     

Tyrosine kinase regulatory action on ileal muscarinic effects of IFN-gamma.

Interferon-gamma (IFN-gamma) is a pleiotropic cytokine that has a large number of immunologic and nonimmunologic functions. We have described that IFN-gamma could activate muscarinic cholinergic receptors (mAchR) of rat intestine, stimulating ileal motility. We also observed that mAchR activation induced inhibition of cAMP levels and stimulation of cGMP formation. The objectives of our work were to clarify the signal transduction pathways involved in regulation of ileal motility through mAchR activation by IFN-gamma. Our results demonstrate that this cytokine produces an ileal cholinergic response through tyrosine kinase activity. The activation of tyrosine kinase mediates ileal contractility, phosphoinositide hydrolysis by phospholipase C, nitric oxide synthase via protein kinase C, and cGMP synthesis. The increment in ileal motility is probably due to hyperproduction of prostaglandin E2 (PGE2) by ileal tissue. This prostanoid is an important mediator because it stimulates ileal motility. We conclude that IFN-gamma not only immunomodulates the gut microenvironment but also exerts a local nonimmunologic regulation on intestinal motility.     

The role of complement in the induction of antibody responses.

To determine the effect of complement on the normal antibody response to T cell-dependent antigens, we immunized normal and C4 deficient guinea-pigs with bacteriophage phi X 174. Following primary immunization with a standard dose (2 X 10(9) PFU/Kg) given intravenously. C4 deficient guinea-pigs produced less antibody than normal guinea-pigs and were unable to maintain measurable antibody levels. Following secondary immunization, antigen clearance of C4 deficient guinea-pigs was delayed and the subsequent antibody response was identical to their primary response without amplification or isotype switch. Increased antigen dose and administration of antigen in adjuvants into footpads improved the responses but did not make them normal. The primary and secondary responses became essentially normal, however, when small amounts of normal guinea-pig serum were given to the deficient animals at the time of the primary (but not the secondary) immunization. We postulate that the contribution of complement to the mature humoral immune response is related to activation of C3. Our data show that antigen initiates a primary immune response. The resultant antigen-antibody complexes interact with complement and are then non-specifically trapped by C3 receptors on dendritic cells, B cells and macrophages. Thus, antigen is selectively accumulated within the lymphoid organs and in turn 'captures' antigen specific B cells by interaction of the trapped antigen with antigen specific sIg. The approximation of specific lymphoid cells, macrophages and antigen permits generation of specific memory cells and ensures prompt, mature antibody response on subsequent antigen exposure.