Interaction of Plasmodium falciparum histidine-rich protein II with human lymphocytes leads to suppression of proliferation, IFN-γ release, and CD69 expression

The presence of histidine-rich protein II (HRP II) synthesized by Plasmodium falciparum in the plasma of malaria patients for longer periods even after parasite clearance raises questions about its extracellular functions. The present study was carried out to examine its influence on host immune system. Recombinant HRP-II protein was radiolabeled with ¹²⁵I to study the specific binding with T and B cells. We found that the binding of ¹²⁵I-HRP II with human T and B cells was specific, concentration dependent, saturable, and reversible. Scatchard plot analysis revealed two classes of binding sites for both T and B cells. For the T cells, the high affinity class had dissociation constant (K _d) of 5.61×10⁻¹¹ M, and the low affinity class had a K _d of 8.58×10⁻¹¹ M. For the B cells, the high and low affinity classes had a K _d of 1.32×10⁻¹¹ and 2.84×10⁻¹¹ M, respectively. Dot-blot, autoradiography, and Western blot analysis also confirmed the specific binding of HRP II with lymphocytes. HRP II significantly inhibited (∼75%) T-cell rosette formation with sheep erythrocytes. HRP II also suppressed proliferation of T and B cells triggered by CD3 and LPS, respectively. We found a reduction in IFN-γ release in T cells preincubated with HRP II. HRP II also reduced the CD69 expression on the T cells. In conclusion, HRP-II binding to human lymphocytes leads to suppression of some of their functions.P. Das and J. S. Grewal contributed equally to this work.     

Tumor-induced myeloid-derived suppressor cell function is independent of IFN-γ and IL-4Rα

Myeloid-derived suppressor cells (MDSCs) are present in most cancer patients and experimental animals where they exert a profound immune suppression and are a significant obstacle to immunotherapy. IFN-γ and IL-4 receptor alpha (IL-4Rα) have been implicated as essential molecules for MDSC development and immunosuppressive function. If IFN-γ and IL-4Rα are critical regulators of MDSCs, then they are potential targets for preventing MDSC accumulation or inhibiting MDSC function. Because data supporting a role for IFN-γ and IL-4Rα are not definitive, we have examined MDSCs induced in IFN-γ-deficient, IFN-γR-deficient, and IL-4Rα-deficient mice carrying three C57BL/6-derived (B16 melanoma, MC38 colon carcinoma, and 3LL lung adenocarcinoma), and three BALB/c-derived (4T1 and TS/A mammary carcinomas, and CT26 colon carcinoma) tumors. We report that although MDSCs express functional IFN-γR and IL-4Rα, and have the potential to signal through the STAT1 and STAT6 pathways, respectively, neither IFN-γ nor IL-4Rα impacts the phenotype, accumulation, or T-cell suppressive potency of MDSCs, although IFN-γ and IL-4Rα modestly alter MDSC-macrophage IL-10 crosstalk. Therefore, neither IFN-γ nor IL-4Rα is a key regulator of MDSCs and targeting these molecules is unlikely to significantly alter MDSC accumulation or function.     

β2-adrenergic receptor agonists modulate human airway smooth muscle cell migration via vasodilator-stimulated phosphoprotein

Severe asthma manifests as airway remodeling and irreversible airway obstruction, in part because of the proliferation and migration of human airway smooth muscle (HASM) cells. We previously reported that cyclic adenosine monophosphate-mobilizing agents, including β(2)-adrenergic receptor (β(2)AR) agonists, which are mainstay of asthma therapy, and prostaglandin E2 (PGE2), inhibit the migration of HASM cells, although the mechanism for this migration remains unknown. Vasodilator-stimulated phosphoprotein (VASP), an anticapping protein, modulates the formation of actin stress fibers during cell motility, and is negatively regulated by protein kinase A (PKA)-specific inhibitory phosphorylation at serine 157 (Ser157). Here, we show that treatment with β(2)AR agonists and PGE2 induces the PKA-dependent phosphorylation of VASP and inhibits the migration of HASM cells. The stable expression of PKA inhibitory peptide and the small interfering (si) RNA-induced depletion of VASP abolish the inhibitory effects of albuterol and PGE2 on the migration of HASM cells. Importantly, prolonged treatment with albuterol prevents the agonist-induced phosphorylation of VASP at Ser157, and reverses the inhibitory effects of albuterol and formoterol, but not PGE2, on the basal and PDGF-induced migration of HASM cells. Collectively, our data demonstrate that β(2)AR agonists selectively inhibit the migration of HASM cells via a β(2)AR/PKA/VASP signaling pathway, and that prolonged treatment with albuterol abolishes the inhibitory effect of β-agonists on the phosphorylation of VASP and migration of HASM cells because of β(2)AR desensitization.     

Differential secretion of TNF-α and IFN-γ by human peripheral blood-derived NK subsets and association with functional maturation

Natural killer cells can be separated into three major subsets (free, binder, and killer) based on their ability to bind and kill sensitive target cells. The nonbinder, nonkiller free cells are the most immature and can be activated to become binders and killers. Natural killer (NK) cells synthesize and secrete several cytokines that are intimately involved in NK activation. This study investigated the secretion of tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) by purified NK cells and NK subsets following activation by various stimuli. K562 target cells stimulated secretion of both TNF- and IFN- by both the binder and the killer subsets but not by the free subset. IFN- activated the secretion of IFN- only, whereas IL-2 activated the secretion of both TNF- and IFN- by the binder and killer subsets and secretion was augmented by the addition of K562 to the cultures. Phorbol myristate acetate (PMA) and ionophore stimulated TNF- and IFN- secretion in both the binder and the killer subsets, though IFN- secretion was more pronounced in the binder subset. Activation of TNF- and IFN- secretion was dependent on de novo protein synthesis. Analysis at the single-cell level demonstrated that the binder subset had the highest frequency of cells secreting IFN-. These results demonstrate that both the binder and the killer subsets can be activated to secrete TNF- and IFN-, whereas the free NK subset secretes little or no TNF- and IFN- following activation. These data suggest that the ability of NK cells to secrete TNF- and IFN- following activation correlates with the functional stage of maturation of NK cells.     

Impairment of lung function might be related to IL-10 and IFN-γ defective production in allergic children

A functional defect of T regulatory cells (Tregs) has been proposed as pathogenic mechanism of allergic reaction. Impairment of lung function frequently occurs in children with respiratory allergy. This study aimed at investigating the possible role of IL-10 and IFN-γ on lung function deterioration in allergic children. Forty children with mild asthma, monosensitized to house dust mites, were evaluated and followed-up for 2 years. Spirometry was performed in all children. IL-10 and IFN-γ were evaluated in in vitro experiments. FEV(1), FVC, and FEF(25-75), evaluated as percent of predicted, significantly diminished over time (p<0.0001, p=0.03, and p<0.0001 respectively). There was a strong relationship between changes in spirometric parameters and IL-10 production and between changes in FEV(1) values and IFN-γ production over time. This preliminary study provided evidence that IL-10 and IFN-γ production could be defective in allergic children prone to develop functional impairment.     

IFN-γ induces IFN-α and IFN-β expressions in cultured rat intestinal mucosa microvascular endothelial cells

Although researchers have recently begun to pay more attention to the immunological characteristics of microvascular endothelial cells (MVECs), there are no reports on whether activation of MVECs by interferon-γ (IFN-γ) exerts any influence on the expressions of IFN-α/β. In the present study, we examined the influence of IFN-γ on the expressions of IFN-α/β in rat intestinal mucous MVECs (RIMMVECs). Different concentrations of IFN-γ were used to stimulate cultured RIMMVECs in vitro, and the cells and cell supernatants were collected at different time intervals. The influence of IFN-γ on the expressions of IFN-α/β in the RIMMVECs was examined at the mRNA and protein levels by real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The results indicated that IFN-γ was able to activate RIMMVECs, thereby leading to upregulated expressions of IFN-α/β. The real-time quantitative PCR analyses indicated that the IFN-α/β mRNA expression levels in RIMMVECs achieved their peak values after stimulation with IFN-γ at 20?ng/mL for 6?h and were increased by 14.88- and 3.82-fold, respectively, when compared with the levels in negative control cells. The ELISA analyses revealed that the IFN-α/β protein expression levels achieved their peak values after stimulation with IFN-γ at 40?ng/mL. The expression of IFN-α protein achieved its peak value at 12?h, while the expression of IFN-β protein achieved its peak value after 6?h. The present results suggest that the expression and secretion of IFNs may participate in the immunologic barrier function of MVECs.     

Interaction of IgE with rat basophilic leukemia cells—v. Binding properties of cell free particles

Particulate material from sonicated rat basophilic leukemia cells was isolated and examined for its ¹²⁵I-IgE binding properties by a rapid filter assay. The binding was saturable and was stoichiometrically inhibited with unlabeled IgE. Binding parameters (at 37°C) were similar to those observed with intact cells though the forward rate constant was up to 2 × higher (2 × 10⁵ M⁻¹sec⁻¹ while the backward rate constant was 2–3 × smaller (∼5 × 10⁻⁶ sec⁻¹) yielding an estimated K_a of ∼4 × 10¹⁰M⁻¹. The binding activity was irreversibly lost after brief exposure to temperatures above 50° and at pH's above 9 or below 5. The activity was largely resistant to proteolytic enzymes and neuraminidase, but not to phospholipase C. Non-ionic detergents led to apparent disruption of the particles.     

IL-36α induces maturation of Th1-inducing human MDDC and synergises with IFN-γ to induce high surface expression of CD14 and CD11c

We show that IL-36α induced maturation of human MDDCs and stimulated differentiation of IFN-γ producing (Type 1) CD3+ lymphocytes but was not as effective as IL-36β in doing so. For the first time, we also show that IL-36α induced expression of CD14 by MDDCs and this was highly potentiated by co-cultured with IFN-γ. In contrast, lipopolysaccharide (LPS) did not increase CD14 expression by MDDCs, suggesting that if MDDCs represent a physiologically relevant population in vivo, they need to be stimulated by relevant inflammatory cytokines prior to CD14 expression and detection of LPS, expressed by Gram negative bacteria. IFN-γ synergised with IL-36α to restore the high levels of CD11c expression by MDDCs, which was reduced by culture with these cytokines in isolation. IL-36α/IFN-γ synergy also correlated with increased binding of the opsonic complement protein (iC3b) to MDDCs. However although IL-36α increased the phagocytic capacity of MDDCs for Salmonella Typhimurium 4/74 this was not synergistically increased by IFN-γ (P > 0.05).In conclusion we report the hitherto unknown effects of IL-36α on the innate cell function of human MDDCs.     

WISH cell line: from the antiviral system to a novel reporter gene assay to test the potency of human IFN-α and IFN-β

Interferons (IFNs) are potent biologically active proteins that are widely used as biopharmaceuticals, so their potency must be correctly identified. Usually, the biological activity is quantified by a bioassay based on its capacity to induce an antiviral state in target cells, but this type of assays is subject to virus manipulation-related issues and they show considerable intra- and inter-assay variability. In this work, we generated a reporter gene assay (RGA) supported on the WISH-Mx/eGFP reporter cell line to determine human type I IFN activity. WISH cells were stably transfected with the enhanced green fluorescent protein (eGFP) gene under the control of type I IFN-inducible Mx2 promoter. This system implies the use of a standardized cell line for human IFN-potency analysis such as WISH cells and the simultaneous use of the sensitive reporter gene eGFP, having also several advantages when compared to antiviral activity assays and other RGAs: it can determine the potency of hIFN-α and hIFN-β using only one cell line showing the highest expression of eGFP after 28h and being only observed in cells treated with type I IFNs due to the specificity of the Mx promoter. It is a sensitive assay and it represents a safe alternative when compared with the conventional antiviral tests. The cell line showed the same sensitivity along 57 generations, allowing its use during two months of successive culture. The inter- and intra-assay coefficients of variation were lower than 20%, demonstrating its reproducibility. In addition, this reporter cell line can be used for the conventional antiviral assay, either for hIFN-α or hIFN-β. In conclusion, we have developed an alternative reporter system for the analysis of type I IFNs, in which its performance make it a suitable candidate to replace or complement conventional bioassays that are currently employed to measure IFN potency.     

Effects of interferon-γ on cell differentiation and cytokine production of a human monoblast cell line,U937

The U937 cell, a human monoblast cell line, has been used as a model to study the function of human monocytes. We investigated the effects of interferon- (IFN-) on Superoxide anion (O ₂ ^– ) production, cell surface antigens, and cytokine production of U937 cells. IFN- treatment enhanced O ₂ ^– production of fMLP or PMA-stimulated U937 cells. IFN- increased the ratio of CD23 and CD 11b positive cells. The fluorescence intensity of CD14 and CD25 was enhanced by IFN- treatment. U937 cells produced IL-1, IL-1, IL-6, and TNF- by lipopolysaccharide (LPS) stimulation. IFN- treatment enhanced TNF- production, but decreased IL-6 production. These results suggest that IFN- differentiates U937 cells to monocytelike cells and it regulates the production systems of IL-6 and TNF- separately in U937 cells.     

Co-operative action of interleukin-10 and interferon-γ to regulate dendritic cell functions

Interleukin-10 (IL-10) and interferon-γ (IFN-γ) double producer is found in a subpopulation of T regulatory type 1 (Tr1) and T helper type 1 (Th1) cells. Consequently, it is of interest how IL-10 and IFN-γ influence the immune system. However, few studies have addressed the co-operative action of these ‘immunosuppressive’ and ‘immunostimulatory’ cytokines. Here, we examine the effect of IL-10 combined with IFN-γ on dendritic cell (DC) functions. Murine bone marrow-derived conventional DCs were stimulated with IL-10 and/or IFN-γ for 24 hr. Tumour necrosis factor-α and IL-12 p40 production by DCs treated with both IL-10 and IFN-γ was significantly lower than that by DCs treated with IL-10 or IFN-γ alone. Major histocompatibility complex class II expression on DCs treated with both cytokines was attenuated compared with that on DCs treated with either cytokine alone. In contrast, levels of inducible nitric oxide synthase and indoleamine 2,3-dioxygenase, which appear to suppress T-cell responses and promote tolerance, in DCs treated with both cytokines were higher than those in DCs treated with IL-10 or IFN-γ alone. Simultaneous treatment with IL-10 and IFN-γ significantly suppressed the ability of DCs to activate CD4⁺ T cells compared with treatment with either cytokine. Therefore, IL-10 and IFN-γ co-operatively suppress the immunostimulatory functions of DCs.     

Molecular Mechanisms of IFN-γ to Up-Regulate MHC Class I Antigen Processing and Presentation

IFN-γ up-regulates MHC class I expression and antigen processing and presentation on cells, since IFN-γ can induce multiple gene expressions that are related to MHC class I antigen processing and presentation. MHC class I antigen presentation-associated gene expression is initiated by IRF-1. IRF-1 expression is initiated by phosphorylated STAT1. IFN-γ binds to IFN receptors, and then activates JAK1/JAK2/STAT1 signal transduction via phosphorylation of JAK and STAT1 in cells. IFN-γ up-regulates MHC class I antigen presentation via activation of JAK/STAT1 signal transduction pathway. Mechanisms of IFN-γ to enhance MHC class I antigen processing and presentation were summarized in this literature review.     

Interaction of MT1-MMP and laminin-5γ2 chain correlates with metastasis and invasiveness in human esophageal squamous cell carcinoma

To gain insights into metastatic mechanisms in esophageal squamous cell carcinoma (ESCC), we established sublines (MLuB1 and MLuC1) with different capacity of spontaneous lung metastasis by subcutaneous injection of a human ESCC cell line (EC 9706) into nude mices. The incidence of the mice with lung metastasis produced by MLuC1 (87%) was significantly higher than that of MLuB1 (22%). The gene expression profiles of the two sublines were compared with cDNA arrays containing 5,000 known genes, and 47 genes were differentially expressed ≥2.0 fold. Laminin-5γ2 chain (Ln-5γ2) was one of the up-regulated genes in MLuC1 cells. Proteolytically processed forms of γ2 are known to promote migration of a multitude of epithelial cells in vitro. Western-blotting analysis revealed that degraded fragments of Ln-5γ2 and active form of membrane-type matrix metalloproteinase-1 (MT1-MMP) in MLuC1 was significantly higher than those in MLuB1. Expression of MT1-MMP was observed in 60 of 75 Ln-5γ2-positive carcinoma tissues (80%). Co-expression of the two proteins was significantly associated with depth of invasion (P = 0.012). Moreover, proteolytic fragments of Ln-5γ2 and active forms of MT1-MMP were frequently found in tumor tissues, whereas in the corresponding normal esophageal tissues there were only intact forms of γ2 and MT1-MMP. siRNA-mediated silencing of MT1-MMP significantly reduced production of γ2′ and γ2x in MLuC1 cells and inhibited cell migration. The results suggest that MT1-MMP is an enzyme responsible for Ln-5γ2 cleavage in ESCC, and interaction between them may play a critical role in promoting invasion and metastasis of human ESCC. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of amino acid substitutions in the human IFN-γR2 on IFN-γ responsiveness

Patients with interferon-γ receptor (IFN-γR) null mutations have severe infections with poorly pathogenic Mycobacteria. The IFN-γR complex involves two IFN-γR1 and two IFN-γR2 chains, in which several amino acid substitutions, some linked to disease and some apparently naturally occurring, have been described. We developed a model system to study functional effects of genetic variations in IFN-γR2. We retrovirally transduced wild-type IFN-γR2 and IFN-γR2 carrying presently known amino acid substitutions in various human cell lines, and next determined the IFN-γR2 expression pattern as well as IFN-γ responsiveness. We determined that the T58R, Q64R, E147K and K182E variants of IFN-γR2 are fully functional, although the Q64R variant may be expressed higher on the cell membrane. The R114C, T168N and G227R variants were identified in patients that had disseminated infections with non-tuberculous Mycobacteria. Of these genetic variants, T168N was confirmed to be completely non-functional, whereas the novel variant G227R, and the previously reported R114C, were partial functional. The impaired IFN-γ responsiveness of R114C and G227R is mainly due to reduced receptor function, although expression on the cell membrane is reduced as well. We conclude that the T58R, Q64R, E147K and K182E variants are polymorphisms, whereas the R114C, T168N and G227R constitute mutations associated with disease.