Interferon-γ Stimulation of Messenger RNA for Human Secretory Component (poly-Ig Receptor) Depends on Continuous Intermediate Protein Synthesis

Secretory component (SC or poly-Ig receptor) plays a key role in mucosal external body fluids. The aim of this study was to elucidate the molecular events underlying IFN-γ-dependent up-regulation of SC. Using a human SC cDNA clone isolated by our laboratory, we found that IFN-γ up-regulated SC mRNA levels in a time- and concentration-dependent manner. Moreover, in situ hybridization showed a striking increase of SC mRNA-positive HT-29 cells after IFN-γ treatment. Inhibition with 5,6-dichloro-l-β-ribofuranosyl-benzimidazole (DRB) indicated a half-life for IFN-γ-induced SC mRNA of approximately 1 h. Cycloheximide (CHX) abolished the IFN-γ-induced accumulation of SC mRNA in a reversible manner; the time-course suggested that de novo synthesis of protein factor(s) with a turnover time shorter than 6 h was required for accumulation of SC message. IFN-γ-stimulated up-regulation of SC expression therefore appears to depend on molecular events similar to those taking place for the activation of several other genes in the Ig supergene family.     

Regulation of Complement Factor H in a Human Liver Cell Line by Interferon-γ

Factor H is a regulatory protein of the alternative pathway of complement activation. The liver is the major site of synthesis. We have used the Hep3b human liver cell line as a model for examining its regulation by interferon-gamma (IFN-gamma). The maximal response was achieved at 50 U/ml of IFN-gamma. An increase in H mRNA was observed as early as 2 h after addition of IFN-gamma; the response peaked at 24 h. The half-life of H mRNA in the presence of IFN-gamma was 3.8 +/- 0.8 h. The increase in H mRNA by IFN-gamma was partly dependent on protein synthesis, as cycloheximide (CHX) reduced the response by 40% and the level of H mRNA decreased in a dose-dependent manner with increasing concentrations of CHX. Phosphorylation events were also important in this induction because the kinase inhibitors staurosporine and genistein inhibited the induction of H mRNA by 88% and 68%, respectively. The induction could be inhibited completely when Hep3b cells were treated with CHX and staurosporine. Thus induction of factor H by IFN-gamma apparently involves two factors. One is likely to be Stat1alpha and the other is a CHX-sensitive protein.     

Fcγ Receptor Heterogeneity in the Human Placenta

Fc receptors for IgG, FcRI (CD64), FcRII (CD32), and FcRIII (CD16) in human placenta were studied by indirect immunohistochemistry using avidin-biotin peroxidase complexes for the staining of cryostat sections. The MoAb 32.2 against FcRI stained cells in the loose connective tissue of the placental villi. The MoAb IV3 (FcRII) and C1KM5 (FcRII) also stained stromal cells and in addition stained the endothelium of the placental villi. The MoAb anti-Leu-11b against FcRIII and B1D6 against a 40-kDa FcR from placenta stained both stromal cells and endothelium as well as the fetal trophoblasts lining the villi. The MoAb 3G8 (FcRIII) also stained trophoblasts and stromal cells but did not stain the endothelium. The heterogeneity of FcR expression on human placenta is established. The function of the different receptors is still unclear.     

Lack of Interferon-γ Receptor does not Influence the Outcome of Infection in Murine Schistosomiasis mansoni

Studies of vaccine-induced immunity in experimental schistosomiasis in mice have suggested that interferon-γ (IFN-γ) is an important factor for the induction of protective immunity against schistosomiasis. The present study compares some parameters during primary schistosome infection in IFN-γ receptor deficient mice and wild type mice. No significant difference in worm burden between the two groups was found. Almost the same number of eggs in the liver as well as typical granulomas with numerous macrophages and eosinophils were observed in both groups of mice. Furthermore, IFN-γ receptor deficient mice infected with S. mansoni displayed a significant reduction in the number of IgG2a secreting cells in the spleen and a significant enhancement of IgA secreting cells in the spleen and in the lungs. These findings suggest that the lack of IFN-γ activity may result in an enhanced dominance of Th2 cells which, however, does not influence the development of a primary schistosome infection.     

Effects of Interferon-α/β and Interferon-γ Preparations on Phagocytosis by Mouse Peritoneal Macrophages

The influence of murine alpha/beta-interferon (Mu IFN-alpha/beta) and murine gamma-interferon (Mu IFN-gamma) preparations on the attachment and ingestion phase of phagocytosis by mouse peritoneal macrophages (MPM) was studied. A non-opsonized strain of Escherichia coli, IgG-opsonized E. coli, and sheep erythrocytes opsonized with IgG (E-IgG) and IgM plus complement factor C3b (E-IgMC) were used as test particles. Pretreatment of MPM with 10(2)-10(3) U/ml of Mu IFN-alpha/beta for 24 h enhanced both attachment and ingestion of bacteria or erythrocytes mediated by the non-specific receptor, the Fc receptor, or the C3b receptor. Higher concentrations had no such effects. In contrast, treatment of MPM with 10(1)-10(2)U/ml of Mu IFN-gamma suppressed attachment and ingestion of non-opsonized and IgG-opsonized E. coli and of E-IgG by 10-40%. Mu IFN-gamma did not influence attachment and ingestion of E-IgMC. The effects were neutralized by specific anti-IFN antiserum. The data indicate that the IFN effect on phagocytic activity is, at least to a large extent, due to modifications of the surface receptors.     

Interleukin-4 and Interferon-γ: The Quintessence of a Mutual Antagonistic Relationship

The two cytokines interleukin (IL)-4 and interferon (IFN)-γ play major roles in the generation and regulation of immune responses. Central in this respect is their mutually antagonistic functions. First, IL-4 promotes T helper cell type 2 (Th2) differentiation and stability and inhibits Th1-cell differentiation. A direct role of IFN-γ in Th1-cell differentiation is debatable, whereas inhibition of Th2-cell differentiation and roles in Th1-cell stabilization are well established functions of IFN-γ. Secondly, IL-4 and IFN-γ also affect antibody class switch and expression of Fc receptors differentially, which strongly affect the effector mechanisms following antibody production. Thirdly, macrophage activities induced or enhanced by IFN-γ, such as expression of certain cytokines, surface molecules and enzymes, are antagonized by IL-4. Together, these functions of IL-4 and IFN-γ place the two cytokines at cardinal positions in the regulation of immune reactions. In this review the known molecular mechanisms underlying the observed functions of IL-4 and IFN-γ are presented and discussed.     

Impaired Mitogen-Induced Interferon-γ Production in Rheumatoid Arthritis and Related Diseases

Peripheral blood lymphocytes from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), Reiter's disease, osteoarthritis, and from healthy volunteers were investigated for interferon-gamma (IFN-gamma) production after mitogen activation. Phytohaemagglutinin stimulation revealed an impaired IFN-gamma production in RA, SLE, and PSS but normal levels in Reiter's disease and osteoarthritis. In RA this deficiency was also seen after pokeweed mitogen, OKT3, and concanavalin A activation. No major differences were found in interleukin 2 (IL-2) production and cell proliferation. The IL-2 receptor expression was reduced on stimulated RA lymphocytes. The deficient IFN-gamma production was compensated in RA by co-stimulation of PHA or OKT3 with phorbol myristic acetate (PMA). In addition, the combination of the calcium ionophore A 23187 and PMA induced a strong IFN-gamma secretion in all patient groups and in the controls.     

Interferon-γ and pulmonary macrophages contribute to the mechanisms underlying prolonged airway hyperresponsiveness

Background Airway hyperresponsiveness (AHR) in asthmatics includes a variable component that persists following an allergen challenge. This may be dissociated from inflammatory cell recruitment, implying a role for resident pulmonary cells in regulating the response. Objective Using improved methods of assessing AHR in a mouse model of allergic airway disease, to investigate the basis of the development of prolonged AHR. Method BALB/c mice were systemically sensitized and then challenged with aerosolized ovalbumin (OVA). Airway and tissue responsiveness were measured at baseline and at 1 day, and 1, 2 and 3 weeks after the last OVA challenge. Inflammatory cell numbers in BALF and levels of mRNA for eotaxin‐1 and ‐2, IFN‐γ, IL‐5 and ‐13 in the lung were measured at each time‐point. In further experiments, the roles of IFN‐γ and of CCR3⁺ and CD4⁺ cells in the development of prolonged AHR were assessed by blockade or depletion with monoclonal antibodies. The role of pulmonary macrophages was assessed by selective chemical depletion of these cells. Results Airway responsiveness was increased above baseline at 1 day after the last OVA challenge, and this was sustained for 1 week. In contrast, tissue‐specific responsiveness was only significantly increased above baseline at 1 day. Development of prolonged AHR was inhibited by neutralization of IFN‐γ or by depletion of pulmonary macrophages, but not by depletion of either CD4⁺ T cells or CCR3⁺ eosinophils. Conclusion An interaction between IFN‐γ and pulmonary macrophages contributed to the prolongation of airway hyperresponsiveness. In contrast, T cells and eosinophils did not contribute to prolongation of AHR. These findings emphasize the importance of the innate host response in the development of manifestations of asthma, as well as its potential relevance as a target for therapeutic intervention. Cite this as: M. Yang, R. K. Kumar and P. S. Foster, Clinical & Experimental Allergy, 2010 (40) 163–173.     

Interferon-γ Enhancement of E-Selectin Expression on Endothelial Cells is Inhibited by Monensin

The expression of E-selectin reaches a maximum 4–6 h after stimulation of human umbilical vein endothelial cells (HUVEC) in vitro with tumour necrosis factor-α (TNF-α) and then declines to basal level within 24 h. If interferon-γ (IFN-γ) is added to the cell culture medium together with TNF-α the surface expression of E-selectin is augmented and prolonged in a synergistic way. The aim of the present study was to investigate if altered protein glycosylation could explain the IFN-γ induced persistent surface expression of E-selectin. SDS–PAGE analysis of HUVEC glycoproteins, metabolically radiolabelled in the carbohydrate portion, indicated that addition of IFN-γ produced an altered protein glycosylation. Lectin blot analysis using the Sambucus nigra agglutinin lectin also indicated differences in protein glycosylation when HUVEC were incubated with IFN-γ/TNF-α compared to TNF-α alone. The kinetics of surface expression of E-selectin were measured using a cell ELISA assay. When HUVEC were incubated with monensin, a potent inhibitor of late Golgi function, together with both TNF-α and IFN-γ, the additive effect of IFN-γ on E-selectin expression was almost abolished. Since monensin is known to affect glycosylation processing, this experiment suggested that the IFN-γ induced change in protein glycosylation might induce the prolonged surface expression of E-selectin. However, when HUVEC were cultured with IFN-γ/TNF-α in the presence of several different inhibitors of N -glycosylation processing, no significant effect of E-selectin expression was observed. Regulation of adhesion molecule expression after activation of endothelial cells is likely to play a pivotal role for the inflammatory response. Further studies are needed to understand the mechanisms underlying this regulation.     

Lyme Neuroborreliosis: Evidence for Persistent Up-Regulation of Borrelia Burgdorferi-Reactive Cells Secreting Interferon-γ

The T-cell response to the aetiologic pathogen Borrelia (B.) burgdorferi in patients with Lyme neuroborreliosis (LN) and in control patients with other neurological diseases was examined by enumerating B. burgdorferi -reactive T cells secreting interferon-γ (IFN-γ) with an ELIspot assay. LN patients had elevated numbers of B. burgdorferi -reactive IFN-γ secreting cells in blood and approximately 20-fold enriched in the cerebrospinal fluid (CSF). A positive correlation existed in CSF between B. burgdorferi -reactive IFN-γ secreting cells and B cells secreting anti- B. burgdorferi IgG antibodies. The up-regulation of antigen-specific IFN-γ secreting cells persisted in peripheral blood up to at least 9 months and in the CSF for at least 4 months after termination of treatment with antibiotics, when the patients were mostly free from clinical signs and symptoms due to LN. How IFN-_γ interplays with other cytokines and influences the pathogenesis of LN remains to be studied.     

Toll-Like Receptor Expression and Function in Subsets of Human γδ T Lymphocytes

Two subsets of human γδ T cells can be identified by T cell receptor (TCR) V gene usage. Vδ2Vγ9 T cells dominate in peripheral blood and recognize microbe- or tumour-derived phosphoantigens. Vδ1 T cells are abundant in mucosal tissue and recognize stress-induced MHC-related molecules. Toll-like receptors (TLRs) are known to co-stimulate interferon-γ (IFN-γ) production in peripheral blood γδ T cells and in Vδ2Vγ9 T cell lines. By microarray analysis, we have identified a range of genes differentially regulated in freshly isolated γδ T cells by TCR versus TCR plus TLR3 stimulation. Furthermore, we have investigated TLR expression in freshly isolated Vδ1 and Vδ2 subsets and cytokine/chemokine production in response to TLR1/2/6, 3 and 5 ligands. TLR1,2,6,7 RNA was abundantly expressed in both subsets, whereas TLR3 RNA was present at low levels, and TLR5 and 8 RNA only marginally in both subsets. Despite abundant RNA expression, TLR1 was rarely detectable by flow cytometry. In contrast, TLR2 and TLR6 proteins were detected in purified Vδ1 and Vδ2 T cells, and TLR3 protein was detected intracellularly in both subsets. TLR1/2/6, 3 and 5 ligands co-stimulated the IFN-γ and chemokine secretion in TCR-activated Vδ1 and Vδ2 subsets, although the levels of IFN-γ secreted by Vδ1 T cells were much lower than those produced by Vδ2 T cells. Our results reveal comparable expression of TLRs and functional responses to TLR ligands in freshly isolated Vδ1 and Vδ2 T cells and underscore the intrinsically different capacity for IFN-γ secretion of Vδ1 versus Vδ2 T cells.     

Tumour Growth Causes Suppression of Autoreactive T-Cell Proliferation by Disrupting Macrophage Responsiveness to Interferon-γ

Normal immune homeostasis is regulated partly by a small population of CD4⁺ T cells that react to autologous major histocompatibility complex class-II molecules on self-cells. Decreased autoreactive T-cell responses are associated with cancer. Tumour growth causes syngeneic macrophages (Mø) to suppress autoreactive T-cell proliferation by decreasing Mø class-II expression and increasing Mø production of the suppressor molecule prostaglandin E₂ (PGE₂). Because interferon-γ (IFN-γ) is a potent Mø activation molecule which regulates both Mø PGE₂ and class-II expression, the effects of IFN-γ on tumour-induced suppression of autoreactive T-cell proliferation were investigated. Exogenous IFN-γ increased normal host (NH) CD4⁺ autoreactive T-cell proliferation stimulated by syngeneic NHMø but decreased proliferation stimulated by tumour-bearing host (TBH) Mø. Antibody (Ab) neutralization of endogenous IFN-γ activity reduced TBH Mø-mediated suppression. Kinetic studies showed that endogenous IFN-γ suppressor activity was not exclusive during T-cell activation. Indomelhacin treatment blocked IFN-γ-induced suppression in TBH Mø-T cell cultures. TBH Mø-T cell cultures contained significantly more PGE₂ than those containing NH Mø. Exogenous IFN-γ increased early PGE₂ production in TBH Mø cultures but decreased production in NHMø cultures. The Ab-mediated neutralization of endogenous transforming growth factor-β or tumour necrosis factor-x reduced TBH Mμ-mediated suppression and blocked IFN-γ-induced suppression. Short-term treatment of Mμ with IFN-γ before their addition to T cells caused TBH Mμ to stimulate T-cell proliferation, which suggests that early suppressor molecule production by TBHMø inhibits synthesis or activity of IFN-γ-induced stimulatory monokines. These results show that tumour growth causes Mø to suppress autoreactive T-cell responses by allowing IFN-γ to induce Mø suppressor molecules, which block production or activity of stimulatory monokines.     

The Influence of Tumour Necrosis Factor-γ, Interleukin-1β and Interferon-γ on the Expression and Function of the Complement Regulatory Protein CD59 on the Human Colonic Adenocarcinoma Cell Line HT29

CD59 is a 18–25kDa glycoprotein which, by inhibiting the formation of the membrane attack complex, protects homologous cells from complement mediated damage. We have described recently the expression and complement regulatory function of CD59 on colonic adenocarcinoma cells both in vivo and in vitro. In this study we have examined the influence of cytokines on the expression and complement regulatory function of CD59 on the colonic adenocarcinoma cell line HT29. CD59 expression on the HT29 cells was up–regulated after stimulation by mononuclear cells activated by mixed lymphocyte reaction and by culture supernatants from activated mononuclear cells. Similarly, a dose–dependent increase in CD59 expression was observed after stimulation with both tumour necrosis factor-γ and interleukin-1β. A dose–dependent increase in the level of CD59 expression was also seen using low concentrations of interferon-γ (IFN-γ), while CD59 expression on cells cultured with high IFN-γ concentrations was comparable to non–stimulated cells. Cytokine treated cells were more resistant to lysis by homologous complement than non–stimulated cells, and the increase in CD59 expression was shown to be partially responsible for this. The present data strengthen the role of CD59 as a possible participant in tumour escape.