Salmeterol inhibits interferon-γ and interleukin-4 production by human peripheral blood mononuclear cells

T-lymphocytes play an important role in allergic asthma. In the present study, the effect of β₂ adrenoceptor agonists was examined on proliferation, interleukin-4 (IL-4) and interferon-γ (IFN-γ) production by human peripheral blood mononuclear cells (PBMC). The proliferation after 24 h phytohaemagglutinin (PHA) activation was significantly inhibited at high concentrations of salmeterol, isoprenaline and salbutamol (≥10⁻⁶M). A U-shaped concentration response curve was observed for the effect of all agonists on IL-4 production 24 h after PHA activation. Maximal inhibition occurred at 10⁻⁹M and amounted to 71% (P<0.02), 38% (P<0.01) and 49% (P<0.01) for salmeterol, isoprenaline and salbutamol, respectively. In contrast, no significant effect of salmeterol (10⁻¹¹-10⁻⁵ M) on IL-4 production could be detected after 96 h. A biphasic concentration response curve was observed for the inhibitory activity of all β-adrenoceptor agonists on IFN-γ production by PBMC 24 h after PHA activation. The first phase reached a plateau at 10⁻⁹ M and the inhibition amounted to 50% (P<0.05), 33% (P<0.01) and 44% (P<0.05) for salmeterol, isoprenaline and salbutamol, respectively. At higher concentrations of the three β-adrenoceptor agonists the inhibition was increased up to 80% (P<0.05), 60% (P<0.05) and 58% (P<0.01), respectively. Similar to the results obtained after 24 h, IFN-γ production after 96 h was biphasically inhibited by salmeterol, and this inhibition (60%) was significant at 10⁻⁵ M. Together, the present data provide clear evidence for concentration-dependent effects of β-adrenoceptor agonists on the IL-4 and IFN-γ production by human PBMC. These results suggest that β-agonists, at low concentrations, predominantly inhibit IL-4 production and may therefore act as anti-inflammatory drugs in allergic asthma.     

Effect of IFN-γ and IL-4 levels on the expression of Fas and Bcl-2 in peripheral blood lymphocytes in hemodialysis patients

To study the correlation between the levels of IFN-γ and IL-4 and the expression of Fas and Bcl-2 in peripheral blood lymphocytes (PBL) in hemodialysis patients, the indirect immune fluorescein labeling method of flow cytometry and solid sandwich enzyme-linked immunosorbent assay were performed for detecting the expression of Fas and Bcl-2 in PBL and the levels of IFN-γ and IL-4 in the serum of 30 hemodialysis patients, respectively. It was found that the expression of Fas in PBL and the level of IL-4 in the serum of hemodialysis patients were significantly higher (P < 0.01), whereas Bcl-2 in PBL and IFN-γ in the serum were significantly lower (P < 0.01) than those of the normal controls. According to statistical analysis, the expression of Fas in PBL had a negative correlation with the level of IFN-γ, but a positive correlation with IL-4 in the serum of hemodialysis patients. Contrarily, the expression of Bcl-2 had a positive correlation with IFN-γ, but a negative correlation with IL-4 in the serum of hemodialysis patients. These results suggest that hemodialysis patients have a suppressed secretion of Th1-associated cytokine IFN-γ, but an increased secretion of Th2-associated cytokines IL-4, and these two aspects may play an important role in the abnormal apoptosis of PBL and its accompanying immune deficiency.     

Human β-defensin-2 enhances IFN-γ and IL-10 production and suppresses IL-17 production in T cells

Psoriasis is an inflammatory dermatosis with enhanced expression of hBD-2 in keratinocytes and infiltration of cytokine-producing T cells, which in turn, up- or down-regulate hBD-2 expression. We determined the serum levels of hBD-2 and cytokines in psoriasis patients and analyzed the effects of hBD-2 on cytokine production in human peripheral blood T cells. Serum hBD-2 levels in patients were higher than those in controls and correlated with PASI, serum IFN-γ, and IL-10 levels and correlated inversely with serum IL-17 levels. IFN-γ, IL-17, IL-22, TNF-α, IL-1β, and IL-6 enhanced, and IL-10, IL-4, and IL-13 suppressed hBD-2 secretion from keratinocytes. hBD-2 enhanced secretion and mRNA levels of IFN-γ, TNF-α, IL-10, IL-1β, IL-6, and IL-22 and reduced those of IL-17 in CD3/28-stimulated T cells. These effects of hBD-2 were counteracted by PTX. hBD-2 induced phosphorylation of JNK, ERK, and Akt in T cells. Inhibitors of these signals attenuated hBD-2-induced production of IFN-γ, TNF-α, IL-10, IL-1β, IL-6, and IL-22. hBD-2 suppressed phosphorylation of STAT3 and enhanced expression of SOCS3 in CD3/28-stimulated T cells. siRNA against SOCS3 reversed hBD-2-induced suppression of IL-17 production and STAT3 phosphorylation. JNK and MEK inhibitors suppressed hBD-2-induced expression of SOCS3. In conclusion, hBD-2 may bind PTX-sensitive GPCR(s) on T cells and act as a stimulator by enhancing IFN-γ, TNF-α, IL-1β, IL-6, and IL-22 production via JNK, MEK/ERK, and PI3K/Akt and as a regulator by suppressing IL-17 production via SOCS3 or by stimulating IL-10 production.     

Production of IFN-γ by CD4(+) T cells in response to malaria antigens is IL-2 dependent

T-cell immune responses are critical for protection of the host and for disease pathogenesis during infection with Plasmodium species. We examined the regulation of CD4(+) T-cell cytokine responses during infection with Plasmodium berghei ANKA (PbA). CD4(+) T cells from PbA-infected mice produced IFN-γ, IL-4 and IL-10 in response to TCR stimulation at levels higher than those from uninfected mice. This altered cytokine response was dependent on parasitemia. To examine the specificity of the response, mice were adoptively transferred with CD4(+) T cells from OT-II TCR transgenic mice and were infected with PbA expressing OVA. Unexpectedly, CD4(+) T cells from the OT-II-transferred wild-type PbA-infected mice showed high levels of IFN-γ production after stimulation with OVA and the cells producing IFN-γ were not OT-II but were host CD4(+) T cells. Further investigation revealed that host CD4(+) T cells produced IFN-γ in response to IL-2 produced by activated OT-II cells. This IFN-γ response was completely inhibited by anti-CD25 mAbs, and this effect was not due to the block of the survival signals provided by IL-2. Furthermore, IFN-γ production by CD4(+) T cells in response to PbA antigens was dependent on IL-2. These findings suggest the importance of IL-2 levels during infection with malaria parasites and indicate that CD4(+) T cells can produce IFN-γ without TCR engagement via a bystander mechanism in response to IL-2 produced by other activated CD4(+) T cells.     

Activation of cord blood myeloid dendritic cells by Trypanosoma cruzi and parasite-specific antibodies,proliferation of CD8+ T cells,and production of IFN-γ

We previously reported that Trypanosoma cruzi, the agent of Chagas disease, induces in congenitally infected fetuses a strong, adult-like parasite-specific CD8⁺ T cell response producing IFN-γ (Hermann et al. in Blood 100:2153–2158, 2002). This suggests that the parasite is able to overcome the immaturity of neonatal antigen presenting cells, an issue which has not been previously addressed. We therefore investigated in vitro the ability of T. cruzi to activate cord blood DCs and compared its effect to that on adult cells. We show that T. cruzi induces phenotypic maturation of cord blood CD11c⁺ myeloid DCs (mDCs), by enhancing surface expression of CD40, CD80, and CD83, and that parasite-specific IgG purified from cord blood of neonates born to T. cruzi-infected mothers amplify such expression. CD83, considered as the best marker of mature DCs, reaches higher level on cord blood than on adult mDCs. Allo-stimulation experiments showed that T. cruzi-activated cord blood mononuclear cells enriched in DCs (eDCs) stimulate proliferation of cord blood and adult CD3⁺ T cells to a similar extent. Of note, T. cruzi-activated eDCs from cord blood trigger more potent proliferation of CD8⁺ than CD8⁻ (mainly CD4⁺) adult T cells, a feature not observed with adult eDCs. T cell proliferation is associated with IFN-γ release and down-regulation of IL-13 production. These data show that T. cruzi potently activates human cord blood mDCs and endows eDCs to trigger CD8⁺ T cell proliferation and favor type 1 immune response. Interestingly, maternal antibodies can strengthen the development of mature DCs that might contribute to overcome the immunological immaturity associated with early life.     

Decrease in interferon-γ production by peripheral blood mononuclear cells in patients with uterine cervical cancer

The interferon- (IFN-) productivity of peripheral blood mononuclear cells (PBMCs) was examined in 30 patients with uterine cervical cancer. The patients under 50 years of age had decreased IFN- production compared with the age-matched controls. The IFN- productivity in the patients over 50 years of age was decreased as well as in the age-matched controls. The proportion of monocytes in PBMCs did not correlate with the IFN- productivity. The prostaglandin E₂ (PGE₂) productivity of PBMCs increased with the progress of cancer. PGE₂ inhibited the IFN- production by PBMCs, and the sensitivity of PBMCs to PGE₂ was increased in the patients and controls over 60 years of age. The addition of indomethacin resulted in an increase in IFN- production by PBMCs. These results suggest that the increased production of PGE₂ and/or increased sensitivity to PGE₂ are responsible for the decreased IFN- production in patients with cervical cancer.     

Impaired IFN-γ production and proliferation of NK cells in multiple sclerosis

NK cells are multicompetent lymphocytes of the innate immune system with a central role in host defense and immune regulation. Studies in experimental animal models of multiple sclerosis (MS) provided evidence for both pathologic and protective effects of NK cells. Humans harbor two functionally distinct NK-cell subsets exerting either predominantly cytotoxic (CD56(dim)CD16(+)) or immunoregulatory (CD56(bright)CD16(-)) functions. We analyzed these two subsets and their functions in the peripheral blood of untreated patients with relapsing-remitting MS compared with healthy blood donors. While ex vivo frequencies of CD56(bright)CD16(-) and CD56(dim)CD16(+) NK cells were similar in patients and controls, we found that cytokine-driven in vitro accumulation and IFN-γ production of CD56(bright)CD16(-) NK cells but not of their CD56(dim)CD16(+) counterparts were substantially diminished in MS. Impaired expansion of CD56(bright)CD16(-) NK cells was cell intrinsic because the observed effects could be reproduced with purified NK cells in an independent cohort of patients and controls. In contrast, cytolytic NK-cell activity toward the human erythromyeloblastoid leukemia cell line K562, the allogeneic CD4(+) T cell line CEM and allogeneic primary CD4(+) T-cell blasts was unchanged. Thus, characteristic functions of CD56(bright)CD16(-) NK cells, namely cytokine-induced NK cell expansion and IFN-γ production, are compromised in the NK cell compartment of MS patients.     

Rosiglitazone,a PPAR-γ agonist,inhibits VEGF secretion by peripheral blood mononuclear cells and ROS production by human leukocytes

Objective  

To investigate the effects of rosiglitazone, a peroxisome proliferator-activated receptor-γ agonist, on the secretion of vascular endothelial growth factor (VEGF) by peripheral blood mononuclear cells (PBMCs) and on the generation of reactive oxygen species (ROS) by leukocytes.     

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.     

Lactobacillus delbrueckii UFV-H2b20 increases IFN-γ production and CD39+CD73+ Treg cell numbers in lungs,and protects mice against experimental allergic asthma

Asthma is a disorder characterized by airflow obstruction, inflammation, declining airway function, bronchial hyperresponsiveness and tissue remodelling. Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”. The use of probiotics is becoming increasingly studied and recent evidence has suggested that it may provide therapeutic benefits in asthma and other diseases. Lactobacillus delbrueckii UFV-H2b20 fulfils all the requirements to be classified as probiotic. Previous studies have already shown the ability of L. delbrueckii UFV-H2b20 to stimulate the immune system. Our objective was to evaluate the protective effects of L. delbrueckii UFV-H2b20 in experimental allergic asthma. We used a murine model of ovalbumin-induced allergic airway inflammation to mimic allergic asthma. Oral treatment with L. delbrueckii UFV-H2b20 improves respiratory parameters and inhibits the inflammatory response in the lungs by decreasing the numbers of inflammatory monocytes, eosinophils and alveolar macrophages, as well as IgE levels. Treatment increased the IFN-γ/IL-4 cytokine ratio. Levels of IL-10 in the lungs were also increased in treated animals. Our results also showed that the probiotic administration increases the number of CD39⁺CD73⁺ T regulatory lymphocytes in the lung, suggesting a role for purinergic signals in the regulation of inflammation promoted by the treatment. Understanding the mechanisms of modulation of the immune system by probiotics could allow the development of probiotic preparations that are safe and have a direct action. Our results suggest that oral administration of L. delbrueckii UFV-H2b20 could be helpful to treat chronic inflammatory airway diseases, such as asthma.     

CD4+CD25+Foxp3+IFN-γ+ human induced T regulatory cells are induced by interferon-γ and suppress alloresponses nonspecifically

Interferon-γ (IFN-γ)-producing CD3(+)CD4(+)CD25(+)Foxp3(+) peripheral blood lymphocytes (PBL) are more frequently detectable in patients with good than in patients with impaired long-term kidney graft function, suggesting an immunoregulatory role of this induced T regulatory (iTreg) subtype. Herein, the in vitro function of separated CD3(+)CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL that were induced by phorbol 12-myristate 13-acetate (PMA)/ionomycin or alloantigenic stimulation was investigated using cell coculture techniques and flow cytometry. CD4(+)CD25(+)Foxp3(+) PBL with intracellular IFN-γ production increased to 26% in cell cultures stimulated with PMA/ionomycin for 6 hours. Recombinant IFN-γ augmented and anti-IFN-γ monoclonal antibody blocked induction of CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL, suggesting their IFN-γ-dependent induction. In addition, CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL produced immunosuppressive interleukin (IL)-10, transforming growth factor-β, and IL-4 intracellularly and expressed both IFN-γ and IFN-γ receptors (CD119) on the cell surface, allowing separation of CD4(+)CD25(+)IFN-γ(+) PBL with 98% purity. Addition of enriched CD4(+)CD25(+)IFN-γ(+) PBL to autologous PMA/ionomycin stimulated PBL decreased blast formation (p < 0.05), indicating suppression of cell proliferation by CD4(+)CD25(+)IFN-γ(+) PBL. CD4(+)CD25(+)IFN-γ(+) PBL separated from primary mixed leukocyte cultures (MLC) and added to autologous or third-party secondary MLC suppressed allogeneic T-cell activation nonspecifically (p < 0.05). We conclude that CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL are induced by IFN-γ, making them sensors for IFN-γ and initial immune responses. Circulating CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL could suppress allogeneic T-cell responses in patients and may be involved in inhibition of the posttransplant alloresponse.     

Methylprednisolone inhibits IFN-γ and IL-17 expression and production by cells infiltrating central nervous system in experimental autoimmune encephalomyelitis

Background  

Glucocorticoids have been shown to be effective in the treatment of autoimmune diseases of the CNS such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the mechanisms and the site of glucocorticoids' actions are still not completely defined. The aim of this study was to investigate the in vivo effect of the synthetic glucocorticoid methylprednisolone (MP) on the expression and production of proinflammatory cytokines interferon (IFN)-γ and interleukin (IL)-17 by cells infiltrating CNS tissue.     

Lack of IL-7 and IL-15 signaling affects interferon-γ production by, more than survival of, small intestinal intraepithelial memory CD8+ T cells

Survival of antigen-specific CD8(+) T cells in peripheral lymphoid organs during viral infection is known to be dependent predominantly on IL-7 and IL-15. However, little is known about a possible influence of tissue environmental factors on this process. To address this question, we studied survival of memory antigen-specific CD8(+) T cells in the small intestine. Here, we show that 2 months after vaccinia virus infection, B8R(20-27) /H2-K(b) tetramer(+) CD8(+) T cells in the small intestinal intraepithelial (SI-IEL) layer are found in mice deficient in IL-15 expression. Moreover, SI-IEL and lamina propria lymphocytes do not express the receptor for IL-7 (IL-7Rα/CD127). In addition, after in vitro stimulation with B8R(20-27) peptide, SI-IEL cells do not produce high amounts of IFN-γ neither at 5 days nor at 2 months postinfection (p.i.). Importantly, the lack of IL-15 was found to shape the functional activity of antigen-specific CD8(+) T cells, by narrowing the CTL avidity repertoire. Taken together, these results reveal that survival factors, as well as the functional activity, of antigen-specific CD8(+) T cells in the SI-IEL compartments may markedly differ from their counterparts in peripheral lymphoid tissues.     

Potent induction of IFN-γ production from cord blood NK cells by the stimulation with single-stranded RNA

Natural killer (NK) cells play important roles in the innate immunity against viral infections. Although newborn infants are more susceptible to severe and recurrent viral infections than adults, the precise role of NK cells in the innate immunity against viral infections during neonatal period is not known. To clarify the functional characteristics of cord blood (CB) NK cells, we examined the capacity of CB NK cells to produce interferon gamma (IFN-γ) in response to the Toll-like receptor (TLR) ligands. We found that NK cells produced a large amount of IFN-γ by the stimulation with ssRNA, a TLR8 ligand, in the presence of interleukin-2 (IL-2), Interferon alpha (INF-α), and monocytes. Surprisingly, CB NK cells produced higher amount of IFN-γ than adult peripheral blood NK cells in this condition. IL-12 produced from monocytes by the stimulation with ssRNA was indispensable for the production of IFN-γ by NK cells. NK cells in cooperation with other innate immune cells may play more important role during the neonatal period than in adults in the host defense against viral infections by high capacity of IFN-γ production to compensate immature acquired immunity.     

Enhanced IL-10 production by CD4+ T cells primed in IL-15Rα-deficient mice

In this study, we investigated the functional outcomes of CD4(+) T cells primed in the absence of IL-15 transpresentation. Compared with their WT counterparts primed in WT mice, IL-15Rα KO CD4(+) T cells primed in KO mice were found to exclusively overproduce IL-10 upon in vitro restimulation(.) The comparable expression of IL-4 and Foxp3 in CD4(+) T cells primed in the WT and IL-15Rα KO mice indicated that this was neither due to T(H) 2- nor Treg cell-differentiation. IL-10 overproduction was also observed when OVA-specific TCR transgenic CD4(+) T (OT-II) cells were primed in KO mice, excluding an intrinsic deficiency of KO CD4(+) T cells. To investigate the WT and KO microenvironment, DCs from both WT and IL-15Rα KO mice were compared. DCs from both backgrounds were indistinguishable in their steady-state survival and in their expression of MHC class II and costimulatory molecules CD80, CD86, and CD40. However, IL-15Rα KO DCs primed OT-II cells in vitro to produce higher levels of IL-10 upon their restimulation. Additionally, IL-15Rα KO DCs produced significantly more IL-10 upon activation, and IL-10 neutralization during DC-mediated in vitro priming abolished IL-10 overproduction by CD4(+) T cells. Thus, IL-15Rα KO DCs provide an IL-10-enriched environment that preferentially primes CD4(+) T cells for more IL-10 production, highlighting a regulatory role for IL-15 transpresentation in CD4(+) T-cell priming.     

Umbelliprenin induced production of IFN-γ and TNF-α, and reduced IL-10, IL-4, Foxp3 and TGF-β in a mouse model of lung cancer

Umbelliprenin is a member of the 7-prenyloxycoumarins with potential therapeutic properties such as cytotoxic effects on various cancer cells. The present study investigates the effect of umbelliprenin on predominance of Th1 and Th2 responses in Lewis lung cancer (LLC) mouse model. The cytotoxic effect of umbelliprenin was explored on LLC cells and mouse splenocytes by MTT assay. Mice into which LLC had been transplanted were treated with umbelliprenin on alternate days, at 2.5?mg/200?µl intraperitoneally. Foxp3, TNF-α and TGF-β mRNA expressions were assessed in tumor and lung tissues of LLC mice. In addition, IL-10, IFN-γ and IL-4 levels were determined in sera and also in splenocyte culture supernatants at the presence of tumor cell lysate (10?µg/ml) and Con A (3?µg/ml) after 72?h. Results showed the cytotoxic effects of umbelliprenin on LLC cells (IC₅₀?=?51.6?±?5.4?µM) while no adverse effect was seen at this concentration on normal splenocytes. TNF-α mRNA expression in both lung and tumor tissues was increased. However, Foxp3 and TGF-β expressions were decreased in tumor tissues. Serum level of IFN-γ was elevated in the umbelliprenin treated cancerous mice compared to the control group while IL-10 and IL-4 secretions were reduced. Tumor size was also decreased in umbelliprenin treated group. In summary, umbelliprenin has shown a partially Th1 bias with a reduction of regulatory immune response. Although the mechanism behind this action is not known, it is speculated that upon changing the Th1/Th2 balance in favour of Th1, umbelliprenin induces its antitumor activity.