IFN-gamma RNA Research Articles

Disparate Role of LIGHT in Organ-specific Donor T Cells Activation and Effector Molecules in MHC Class II Disparate GVHD

The present studies determined the role of LIGHT on organ-specific cytokine and effector molecules in acute graft-versus-host disease. Cytokine and effector molecules were assessed by flow cytometry and quantitative PCR. More CD4+ spleen cells (SpC) expressing interferon-gamma (IFNgamma) and interleukin-2 were noted in SpC isolated from lethally irradiated bm12 X B6 F1 recipients of B6 donor SpC and T cell-depleted bone marrow cells and control Adv-betagal than in transplant (bone marrow transplantation (BMT)) recipients who had received Adv-LTbetaR-Ig or Adv-herpes simplex virus entry mediator (HVEM)-Ig. IFNgamma RNA levels from SpC, small intestines, and large intestines of control BMT recipients were significantly higher than those who had received Adv-HVEM-Ig. Granzyme B levels from SpC and small intestines of control BMT recipients were significantly higher than those that had received the Adv-LTbetaR-Ig. In contrast, BMT recipients of Adv-HVEM-Ig had lower granzyme A levels than controls in their large intestines. LIGHT inhibition differentially affects cytokines and effector molecules in SpC, small intestines, and large intestines, implicating different organ-specific pathways.

Interleukin 21 Enhances the Expansion and Anti-Tumor Cytotoxic Activity of Cytokine-Induced Killer Cells Derived from Both Peripheral Blood and Cord Blood In Vitro.

Interleukin 21(IL-21) is a new member of interleukin 2 cytokine families which was discovered in 2000. IL-21 is produced by activated CD4 positive cells, and is known to influence T, B, NK cells and DC, and has potent anti-tumor effects. For example, IL-21 can improve the proliferation of B lymphocytes, enhance the production of IgG1; improve the proliferation and enhance the anti-tumor activity of both NK and T cells. The cytokine-induced killer (CIK) cells, which are characterized with the phenotype of CD3+CD56+, are the effective cells on adoptive cellular immunotherapy against tumors. We hypothesize that IL-21 could also affect the proliferation and function of CIK cells, thus play a certain role in the anti-tumor immunotherapy by CIK cells. The peripheral blood mononuclear cells (PBMC) and cord blood mononuclear cells (CBMC) from healthy donors were stimulated with anti-CD3 (OKT3) monoclonal antibody and IFNgamma and then expanded with IL-2 and with/without IL-21(200ng/ml). CD3+CD56+ CIK cells were counted by flow cytometry. Net lactate dehydrogenase release from target cells incubated with CIK cells was used as an index of CIK cells cytotoxicity against chronic myeloid leukemia cell line K562 and a variety of tumor target cells from patients. The concentration of the IFNgamma in the culture supernatant was measured by enzyme-linked-immunoassay, the quantity of IFNgamma RNA was measured by RT-PCR assay, and the cytotoxic activity against K562 cells by the culture supernatant was also detected. Cultured with IL-21, at day 14, the quantity of CIK cells was increased from a median of 17.5% to 26.5% (PBMC original) and from 33.8% to 55.9% (CBMC original); The cytotoxic activity rates against K562 cells by CIK cells were increased from 24.0% to 52.2% (PBMC original) and from 35.1% to 79.7% (CBMC original); The concentration of IFNgamma in the culture supernatant was increased for 1.9-fold (PBMC original), and for 3.2-fold (CBMC original); The cytotoxic activity against K562 cells by the culture supernatant was increased for 1.8-fold (PBMC original) and for 2.7-fold (CBMC original); The expression of IFNgamma RNA in CIK cells was also markedly increased derived from both PBMC and CBMC when cultured with IL-21. Moreover, the cytotoxic activity against leukemia cells from 11 patients (6 with acute lymphoblastic leukemia, 5 with acute myeloid leukemia) by CIK cells derived from CBMC were also detected. The cytotoxic activity rates were at a median of 68.3% (range, 34.7%–86.4%) when CIK cells were cultured with IL-21, rates that contrasted drastically to the cytotoxic activity rates when CIK cells were cultured without IL-21, which were only at a median of 37.4% (range, 16.1%–60.0%). In conclusion, our data indicated that IL-21 could enhance the expansion of CIK cells and their anti-tumor activity derived from both PBMC and CBMC in vitro, IFNgamma was evolved in this course although the mechanism still need to be explored. These observations open up the possibility of imagining a future clinical application of IL-21 in the anti-tumor immunotherapy by CIK cells.

Experimental study on the effects of interferon-gamma gene on Tenon's capsule fibroblasts in vitro

To investigate the effects of interferon-gamma (IFN-gamma) gene on Tenon's capsule fibroblasts in vitro. Using lipofectAMINE, IFN-gamma gene was transferred to human Tenon's capsule fibroblasts with plasmid pcDNA3IFN-gamma in vitro. Cells treated with plasmid pcDNA3 without IFN-gamma gene were used as the controls. The transfected fibroblasts were screened by G418. The expression of IFN-gamma was determined by RT-PCR, immunohistochemistry and flow cytometry assay. The effects of IFN-gamma on the proliferation of Tenon's capsule fibroblasts were evaluated by flow cytometry and MTT. The Tenon's capsule fibroblasts transferred the IFN-gamma gene could express the IFN-gamma RNA and protein transiently. The proliferation of the fibroblasts transferred the IFN-gamma gene was inhibited. The proliferation of the Tenon's capsule fibroblasts in vitro can be inhibited by transferred IFN-gamma gene.

Interleukin 18 and associated markers of T helper cell type 1 activity in coeliac disease.

Coeliac disease (CD) is caused by a T helper cell type 1 (Th1) response in the small intestinal mucosa to dietary gluten. Paradoxically, interleukin (IL)-12, the major Th1 inducing factor, is undetectable in the mucosa of active CD. IL-18 is a recently described cytokine capable of promoting T cell interferon (IFN)-gamma production and facilitating Th1 cell polarisation. To examine expression of IL-18 and IL-18-associated Th1 proteins in CD. IL-18 and IFN-gamma RNA transcripts were determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). IL-18 and caspase-1 protein expression were assessed by western blotting. Caspase-1 activity was determined using a commercially available assay. RNA transcripts for the IL-18 receptor subunits, IL-1 receptor related protein (IL-1 Rrp) and accessory protein-like subunit (AcPL), and IL-18 induced Th1 specific T box transcription factor (T-bet) were measured by RT-PCR and Southern blotting. IL-18 RNA transcripts were found in all mucosal samples analysed, with no difference between CD patients and controls. By western blot analysis, a large protein of approximately 24 kDa, corresponding to the immature IL-18, was detected in all mucosal samples from CD patients and controls. In contrast, mature IL-18 was only seen in CD patients. Immunoreactivity corresponding to both immature and mature caspase-1 was present in both CD and control samples. Tissue homogenates from CD patients and controls expressed similar levels of caspase-1 activity. IL-1Rrp and AcPL were seen in all samples but were expressed at greater levels in the mucosa of CD patients. T-bet was also upregulated in CD. Active IL-18 is expressed in CD as well as other markers of Th1 polarisation.

A study of antibody and T cell recognition of rhoptry-associated protein-1 (RAP-1) and RAP-2 recombinant proteins and peptides of Plasmodium falciparum in migrants and residents of the state of Rondonia, Brazil.

Humoral and cellular responses were examined among natives and migrants in an area of the Amazon region of Brazil. Rhoptry-associated protein-1 (RAP-1) and RAP-2 expressed in Escherichia coli expression systems, a peptide corresponding to the epitope bound by inhibitory anti-RAP-1 antibodies, and four other RAP-1 and RAP-2 synthetic peptides were used in these studies. Plasma from the native population had greater IgG reactivity to the N-terminal third of RAP-1 than the migrant population; both populations had low levels of IgM to this region of RAP-1. The IgG reactivity to RAP-2 and to the C-terminal third of RAP-1, as well as for all the peptides, including the peptide from the inhibitory domain, were low or absent in both populations. In contrast, there were a high number of subjects with an IgM response to the peptides. Cellular responses were measured by proliferation of peripheral blood mononuclear cells (PBMC) and, in some subjects, by reverse transcription-polymerase chain reaction for interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-4, and IL-10. Proliferation of PBMC was low when stimulated by recombinant proteins, peptides, or parasite lysate. Both RAP-1 and RAP-2 stimulated cytokine production by donor T cells; IL-2, IL-4, and IFN-gamma RNA transcripts were observed in response to recombinant proteins and parasite lysate, but with no uniform trends. From the observed antibody responses, RAP-1 appears to be more immunogenic than RAP-2.

Novel activation of gamma-interferon in nonimmune cells during human cytomegalovirus replication.

This is the first study documenting the induction of gamma-interferon (IFN-gamma) in human embryonic fibroblasts during human cytomegalovirus (HCMV) replication. Infection of cells with HCMV resulted in the consistent production of IFN-gamma RNA, as determined by RT-PCR and Northern blot analysis. Western blot analysis of cell lysates and immunoprecipitates from the cultural fluids of infected cells demonstrated the presence of IFN-gamma at the protein level. Induction of IFN-gamma required infectious HCMV, since high-dose ultraviolet inactivation of the virus stock eliminated IFN-gamma production. Further, IFN-gamma induction appears to be a late event in the virus replication cycle, since inhibition of HCMV DNA synthesis (e.g., phosphonoacetic acid) blocked the increase in IFN-gamma. Soluble factor(s) released from HCMV-infected cells apparently did not contribute to the induction of IFN-gamma, since virus stocks from which virus had been removed by sedimentation did not induce production of IFN-gamma. The appearance of IFN-gamma at late stages of HCMV infection and its elimination in the presence of an inhibitor (Actinomycin D) of RNA synthesis indicate a true transcriptional induction of this lymphokine at the RNA and protein levels. The significance of IFN-gamma production with regard to the replication and pathogenesis of HCMV in vitro and in vivo will require further investigation.

Interleukin 12 is produced in vivo during endotoxemia and stimulates synthesis of gamma interferon

Gamma interferon (IFN-gamma) is produced in response to circulating lipopolysaccharide (LPS) and contributes to the lethality of endotoxic shock. To address the cellular source of IFN-gamma production in vivo, T cells and B cells were magnetically purified from C57BL/6 mouse spleens 5 h following endotoxin injection. IFN-gamma RNA was abundant in splenic CD4+ and CD8+ T cells and in a T- and B-cell-depleted population of splenocytes containing 34% NK1.1+ natural killer (NK) cells. Because interleukin 12 (IL-12) is a known inducer of IFN-gamma synthesis by cultured T cells and NK cells, we examined whether IL-12 might be involved in IFN-gamma release during endotoxemia. mRNA encoding the p40 subunit of IL-12 increased markedly in the spleens of C57BL/6 mice at 2 h after LPS injection, whereas p35 IL-12 mRNA was constitutively expressed at all times. Bioactive IL-12 (p70 heterodimer) was detected in mouse serum at 2 to 4 h after LPS injection. Similar results were obtained using a p40 subunit-specific enzyme-linked immunosorbent assay. Endotoxin-insensitive C3H/HeJ mice generated threefold less IL-12 p70 and IFN-gamma at these times than endotoxin-sensitive C3H/HeOuJ mice. Pretreatment of mice with polyclonal anti-mouse IL-12 antibody reduced IFN-gamma levels present at 6 h post-LPS nearly sixfold in three separate experiments. These studies support a role for IL-12 as a proximal stimulator of IFN-gamma release during endotoxemia.

Interferon expression in Crohn's disease patients: increased interferon-gamma and -alpha mRNA in the intestinal lamina propria mononuclear cells.

The in vivo interferon (IFN) activation in Crohn's disease was evaluated by measuring the relative amounts of IFN-alpha and -gamma mRNA in freshly isolated human lamina propria mononuclear cells (LPMC) from patients with Crohn's disease and controls. Both IFN-gamma and IFN-alpha mRNA, as estimated by dot blot analysis, were increased in Crohn's disease (LPMC), although the relative amounts of IFN mRNA appeared to differ among patients. Appreciable amounts of IFN-gamma mRNA were found in Crohn's disease peripheral blood mononuclear cells (PBMC) extracts, whereas the same cells were negative for IFN-alpha mRNA. Only minute amounts of IFN-gamma RNA were found sporadically in control LPMC while no IFN-alpha was detected. Control PBMC were shown to be virtually negative for both IFN-alpha and IFN-gamma mRNA. These data suggest that IFN induction in the normal human gut is a well-controlled function and that in Crohn's disease tissues, both IFN-gamma and IFN-alpha production are dysregulated. The increased IFN activity may represent a major feature in the induction and perpetuation of the chronic inflammatory process in Crohn's disease.

Expression of cytokine/growth factors and their receptors in human melanoma and melanocytes

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.

IMPAIRED ACTIVATION OF TUMORICIDAL FUNCTION IN MACROPHAGES FROM MAMMARY-TUMOR BEARERS - THE ROLE OF IFN-GAMMA

The growth of the DI-DMBA-3 mammary adenocarcinoma in BALB/c mice results in a profound deficit in the tumoricidal function of their peritoneal elicited macrophages (PEM) after stimulation with lipopolysaccharide (LPS). The capacity of these macrophages to respond to stronger signals, such as a combination of LPS and low levels (5 U/ml) of interferon-gamma (IFN-gamma) is also impaired. Importantly, a combination of high levels (50 U/ml) of IFN-gamma with 10 mug/ml of LPS, is able to trigger a cytolytic response in macrophages from tumor bearing mice against mammary tumor target cells, indicating that their lytic machinery is intact. However, IFN-gamma production is severely diminished in T lymphocytes from tumor bearing mice as detected by ELISA, moreover, Northern blots revealed that the levels of IFN-gamma RNA are also decreased in T cells from tumor bearers. The cooperation between T cells and macrophages is mediated, at least in part, by IFN-gamma. Thus, mammary tumors have die potential to overcome host defenses, either by affecting the capacity of macrophages to respond adequately to activating agents, and/or by impairing the production of T-cell derived lymphokines important in macrophage activation for tumor killing.

Transfected Leishmania expressing biologically active IFN-gamma.

Infection of susceptible BALB/c mice with Leishmania major leads to progressive infection with the failure to expand and activate Th1 CD4+ T cells that elaborate IFN-gamma, a critically implicated cytokine for control of disease. We used the recently described capacity to express foreign genes in trypanosomatids to introduce into Leishmania the murine IFN-gamma gene on a drug-selectable plasmid under the constitutive control of intergenic tubulin sequences. Several clones of L. major were established and demonstrated to contain IFN-gamma DNA and IFN-gamma RNA that was appropriately trans-spliced with the Leishmania-specific leader sequence, and to secrete IFN-gamma into the media. The secreted IFN-gamma was biologically active as assessed by up-regulation of class II MHC Ag and induction of macrophage nitric oxide synthase activity in a macrophage cell line. Infection of nude mice with IFN-gamma-containing organisms resulted in significantly slower progression of disease as compared to infection with organisms containing the empty plasmid, suggesting that biologically important activation of infected macrophages might be occurring in vivo. Infection of genetically susceptible BALB/c mice, however, did not impede the expansion of Th2 cells and the inexorable progression of disease. Despite the demonstration of increased levels of IFN-gamma transcription in vivo, induction of nitric oxide synthase in macrophages and expression of Ly-6, and IFN-gamma-inducible Ag, on CD4+ lymphocytes could not be shown. In all cases, organisms recovered from tissue amastigotes contained the IFN-gamma plasmid and secreted active IFN-gamma. The data confirm earlier studies that IFN-gamma alone is not sufficient to impede activation and maturation of Th2 cells in susceptible mice, even when targeted directly to the infected cell.

Specific interferon genes are expressed in individual cells in the peritoneum and bone marrow of normal mice.

The use of a highly sensitive method of in situ hybridization capable of detecting one copy of IFN mRNA per cell showed that from 20-50% of the cells from the peritoneum and bone marrow of both normal pathogen-free and axenic mice exhibited grain counts significantly greater than background levels following hybridization with riboprobes specific for the mouse interferon-alpha (IFN-alpha), IFN-beta, or IFN-gamma genes. Labeling was shown to be specific, as the labeled probe was displaced by a 200-fold excess of the specific unlabeled probe but not by a 200-fold excess of an unrelated probe. Grain counts were reduced to background levels when cells were pretreated with ribonuclease prior to in situ hybridization. The extent of labeling with either IFN-alpha or IFN-beta-specific probes increased following i.v. inoculation of mice with the IFN-inducer Newcastle disease virus (NDV) whereas the degree of labeling observed with a probe specific for beta-actin remained unchanged. No significant differences were observed in the number of bone marrow or peritoneal cells that expressed IFN-alpha or IFN-beta mRNA from either high (C57B1/6) or low (BALB/c) IFN-producing strains of mice. The majority of IFN-alpha and IFN-beta-containing cells from both the bone marrow and peritoneum of normal pathogen-free and axenic mice resembled monocytes morphologically, whereas the majority of IFN-gamma mRNA-containing cells resembled small lymphocytes. In addition, in the bone marrow a number of large cells which resembled megacaryocytes were found to express high levels of IFN-alpha mRNA. Nuclear run-on assays showed that IFN-alpha and IFN-beta genes were actively transcribed in both bone marrow and peritoneal cells from normal and axenic mice. Low levels of de novo IFN-gamma RNA synthesis were detected in the nuclei of peritoneal cells only. The expression of IFN genes in individual cells in the tissues of normal animals may constitute a basis for the regulation of both homeostasis and host defense against virus infection and neoplastic cells.

Interleukin 2 and concanavalin A stimulate interferon-gamma production in a murine cytolytic T cell clone by different pathways.

We identified a variant murine cytolytic T lymphocyte (CTL) clone which, in contrast to the parent clone and all other murine T cell populations tested, was found to have acquired spontaneously the ability to produce interferon-gamma (IFN-gamma) in response to recombinant interleukin 2 (rIL-2). IFN-gamma production in response to concanavalin A (Con A), which was characteristic of all T cell populations tested, was preserved in this variant. The IFN produced by the variant in response to either stimulus was active in both a macrophage-activating factor assay and an anti-viral assay. Both activities induced by either stimulus could be blocked by monoclonal anti-IFN-gamma antibodies. Upon Northern blot analysis using an IFN-gamma-specific cDNA probe, the IFN-gamma RNA isolated from variant cells stimulated with Con A or IL-2 were found to migrate equivalently. The unusual pattern of responsiveness in this variant CTL was exploited to compare the mechanisms involved in induction of IFN-gamma production by Con A or IL-2. Striking differences were observed. Unlike IFN-gamma production induced by Con A, IFN-gamma production induced by IL-2 was not accompanied by an elevation of intracellular Ca2+ levels, did not require physiologic extracellular Ca2+ levels, and was not inhibited by the immunosuppressive agent cyclosporin A. Thus, in this variant CTL clone, conditions that have ordinarily been associated in an obligate manner with lymphokine gene expression were found instead to be related to the specific mode of stimulation.

Activation of a human T cell line: a two-stimulus requirement in the pretranslational events involved in the coordinate expression of interleukin 2 and gamma-interferon genes.

In the resting state, the T3-positive, human T cell line Jurkat does not synthesize detectable amounts of either interleukin 2 (IL 2) or gamma-interferon (IFN-gamma). Activation of Jurkat as measured by the secretion of substantial amounts of both lymphokines requires two distinct signals. One signal is produced by the phorbol ester, phorbol myristate acetate, and the other by either phytohemagglutinin or antibodies to T3. To elucidate the molecular events by which these activation signals lead to the synthesis of IL 2 and IFN-gamma activity we used cDNA probes to follow the appearance of IL 2 and IFN-gamma-specific transcripts after activation of Jurkat. These studies demonstrate that both signals are required for the appearance of IL 2 or IFN-gamma-specific transcripts and that the appearance of IL 2 and IFN-gamma RNA is coordinate with regard to a) the signals required for their production, b) the kinetics of their appearance, and c) the inhibition of their appearance by cyclosporin A. These studies suggest that distinct T cell-activation signals may operate through a common regulatory pathway involved in the expression of both IL 2 and IFN-gamma genes.