Levels Of IL-10 In Supernatants Research Articles

Adiponectin/AdipoR1 Axis Promotes IL-10 Release by Human Regulatory T Cells.

BackgroundAdiponectin is an important immunomodulatory mediator in inflammatory conditions. While we previously showed that adiponectin receptor 1 (AdipoR1) is expressed in murine regulatory T cells (Tregs), its expression in human Tregs remain unknown. Here, we examined the expression of AdipoR1 in human Tregs and whether its ligand, globular adiponectin (gAd) affects the Treg ability to secrete IL-10 and the role of Type 2 (T2) inflammation in such process.MethodsHuman Tregs from peripheral blood were analyzed by flow cytometry for AdipoR1, Helios and IL-10 expression. CD4+ T cells enriched from peripheral blood mononuclear cells (PBMCs) were cultured in the presence or the absence of gAd or the chemical adiponectin receptor agonist, AdipoRon, or in a T2 cytokine milieu. Flow cytometry was then used to assess intracellular IL-10, IL-10 secreting cells, FOXP3 and Helios expression, and phosphorylated p38 MAP kinase (MAPK). IL-10 levels in CD4+ T cell supernatants were quantified by ELISA.ResultsWe found that a subset of human Tregs expressed AdipoR1. Importantly, more Helios- cells expressed AdipoR1 than Helios+ cells. Likewise, there was a higher frequency of IL-10+ cells within Helios- AdipoR1+ Tregs compared to Helios+ AdipoR1+ Tregs. In contrast, the IL-10 mean fluorescence intensity (MFI) was higher in Helios+ AdipoR1+ Tregs compared to Helios-AdipoR1+ Tregs. When human CD4+ T cells were treated with gAd or AdipoRon, a significant increase in IL-10 secretion, FOXP3 expression, and p38 MAPK phosphorylation was observed in Helios- AdipoR1+ Tregs. Interestingly, gAd under T2 cytokine milieu significantly increased the intracellular levels of IL-10, mainly in Helios+ AdipoR1+ Tregs, and IL-10 levels in supernatants of CD4+ T cells.ConclusionsCollectively, our findings suggest that adiponectin/AdipoR1 axis promotes IL-10 release by Tregs, mainly in Helios- Tregs, and the effect was amplified by T2 inflammation in Helios+ Tregs.

IL-10-producing B cells play important role in the pathogenesis of recurrent pregnancy loss

Interleukin (IL)-10-producing B cells are recently known for their regulatory function in several disorders. However, the possible role of these cells remains unclear in recurrent pregnancy loss (RPL) pathogenesis. Total B cells from 24 RPL patients with cellular immune abnormalities, as well as that of 25 normal pregnant women were cultured and stimulated by Toll Like Receptor (TLR) agonists (CpG oligodeoxynucleotides (ODN) and imiquimod). Then, the frequency of IL-10+ CD19+ B cells was found out using flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to assess the levels of IL-10 in supernatant medium and serum of stimulated B cells, as well as those of several serum autoantibodies. Real-Time PCR method was carried out for determining the IL-10 expression level and specific genes transcripts. RPL patients indicated a lower proportion of IL-10+ CD19+ B cells, and reduced levels of IL-10 in both serum and supernatant of the culture medium of the stimulated B cells. According to the results, total IgG levels was greater in serum of RPL patients in comparison with healthy pregnant women. Similarly, the percentage of these cells was negatively correlated with serum total IgG levels and the number of miscarriages. The expression levels of the mRNA of programmed death-ligand 1 (PD-L1) and IL-10 were lower in RPL patients, while those of x binding protein 1 (XBP-1), interferon regulatory factor 4 (IRF4), and B lymphocyte-induced maturation protein 1 (BLIMP1) were significantly increased. These observations indicated that the reduction in the population of peripheral blood IL-10-synthesizing B cells may prompt RPL pathogenesis, suggesting suppressive effects of these cells on autoantibody production and successful pregnancy outcomes.

Schistosoma mansoni antigens alter activation markers and cytokine profile in lymphocytes of patients with asthma

Asthma is a chronic disease characterized by airway inflammation, obstruction and hyperresponsiveness. Severe asthma affects a small proportion of subjects but results in most of the morbidity, costs and mortality associated with the disease. Studies have suggested that Schistosoma mansoni infection reduces the severity of asthma and prevent atopy. ObjectiveWe evaluated the ability of S. mansoni antigens, Sm29 and Sm29TSP-2 to modulate lymphocyte activation status in response to the allergen of the mite Dermatophagoides pteronyssinus (Der p1) in cell cultures of individuals with asthma. MethodsThirty four patients were enrolled in this study: seventeen patients with severe asthma (SA group), seventeen patients with mild asthma (MA group) and six controls with no asthma. Peripheral blood mononuclear cells (PBMC) were obtained and stimulated with Sm29 and Sm29TSP-2 in the presence or absence of Der p1. The expression of surface markers and cytokines on lymphocytes was evaluated by flow cytometry and the levels of IL-10 in the culture supernatant were determined by ELISA. ResultsThe addition of Sm29 and Sm29TSP-2 antigens to PBMC cultures from both groups of subjects with asthma stimulated with Der p1 reduced the frequency of CD4+CD25low cells whereas and increased frequency of CD4+CD25high population was observed compared to unstimulated cultures. Moreover, cultures stimulated with Sm29TSP-2 showed a reduction in the frequency of T cells expressing CD69, IFN-γ, TNF and TGF-β in the MA group and an increase in the frequency of CD4+TSLPR+ T cells in the SA group. The addition of Sm29 to the cultures reduced the frequency of CD4+CD69+ and CD4+IL-5+ T cells in all asthmatic groups, and reduced the frequency of CD4+ T cells expressing IL-13 in the MA group. The cultures stimulated with Sm29 and Sm29TSP-2 showed an increase in the level of IL-10 in the supernatants. ConclusionThese results suggest that the addition of Sm29 and Sm29TSP-2 to the cells cultures from subjects with asthma reduced cell activation markers and altered the cytokine production pattern in a way that can potentialy control the inflammatory response associated with asthma.

Modulatory effects of interferon-γ and interleukin-4 on cellular immune responses against Hypoderma lineatum antigens.

This study investigates the in vitro modulatory effects of interferon-γ (IFN-γ) and interleukin-4 (IL-4) on both proliferative bovine T cell responses and IL-10 production induced by different antigens [crude larval extract and the purified fractions hypodermin A, B and C (HyA, HyB, HyC)] obtained from first instars of Hypoderma lineatum (Diptera: Oestridae), alone or in the presence of the mitogen concanavalin A. Incubation with the different parasitic antigens resulted in significant inhibition of T cell proliferation and IL-10 production, which, in general, did not revert after the addition of IFN-γ and IL-4. In the absence of antigens, IL-4 induced significant inhibition of mitogen-induced T cell responses. Exogenous IFN-γ exhibited an inhibitory effect on cell proliferation in the presence of the purified fractions HyB and HyC. These in vitro data suggest that far from neutralizing the effects of larval antigens, the addition of IFN-γ potentiates their anti-proliferative activity; by contrast, IL-4 had no consistent effects on proliferative responses to Hypoderma. IL-4 provoked an increment of IL-10 levels in supernatants of HyB-stimulated cells. In conclusion, exogenous IFN-γ and IL-4 were unable to counteract the suppressor effects of H. lineatum antigens.

Changes of Rho kinase activity in peripheral blood T lymphocytes from patients with atopic dermatitis and their significance

Objective To evaluate changes of Rho kinase (ROK) activity in peripheral blood T lymphocytes from patients with atopic dermatitis (AD), and to analyze their clinical significance. Methods Eight milliliters of heparin-anticoagulated blood samples were collected from 60 patients with AD and 60 healthy human controls followed by separation of T lymphocytes and sera from these blood samples as well as culture of isolated T lymphocytes with 10% fetal bovine serum for 24 hours. Both patient- and control-derived T lymphocytes were classified into two groups to be cultured with patient- or control-derived sera. In addition, some patient-derived T lymphocytes were classified into 4 groups: Y27632 group treated with the Rho kinase-specific inhibitor Y2763, CD3/CD28 group treated with anti-CD3/anti-CD28 monoclonal antibodies, Y27632 + CD3/CD28 group treated with Y27632 and anti-CD3/anti-CD28 monoclonal antibodies, and control group treated with patient-derived sera. Subsequently, Western-blot analysis was performed to evaluate ROK activity in cells, methyl thiazolyl tetrazolium (MTT) assay to evaluate proliferative activity of T lymphocytes, and ELISA to measure interleukin 6 (IL-6) and IL-10 levels in supernatants of T lymphocytes. Results ROK activity was significantly lower in fresh T lymphocytes from patients than in those from healthy controls (2.47% ± 0.89% vs. 0.65% ± 0.35%, t = 2.729, P 0.05). Compared with T lymphocytes cultured with control-derived sera, those cultured with patient-derived sera showed significantly increased ROK activity (F= 8.22, P< 0.001). Concretely speaking, ROK activity was significantly higher in patient-derived T lymphocytes cultured with patient-derived sera than in those cultured with control-derived sera (2.41% ± 0.87% vs. 0.76% ± 0.41%, P< 0.05), and higher in control-derived T lymphocytes cultured with patient-derived sera than in those cultured with control-derived sera (2.17% ± 0.85% vs. 0.64% ± 0.33%, P< 0.05) at 24 hours. Y27632 could significantly inhibit the proliferation of as well as secretion of IL-6 (F = 18.68, 22.95, respectively, both P< 0.001) by patient - derived T lymphocytes, but had insignificant effects on secretion of IL-10. The cellular proliferative activity and IL-6 supernatant level were significantly lower in the Y27632 group than in the control group, and lower in the Y27632 + CD3/CD28 group than in the CD3/CD28 group (all P< 0.05). Conclusion Aberrant activation of ROK exists in T lymphocytes from patients with AD, which may play a certain role in the pathogenesis of AD. Key words: Dermatitis, atopic; T-lymphocytes; rho-Associated kinases; Signal transduction; Interleukin-6; Interleukin-10

Differential T-Helper Cell Polarization after Allergen-Specific Stimulation of Autologous Dendritic Cells in Polysensitized Allergic Patients

Background: Dendritic cells (DCs) play an important role in the induction and regulation of adaptive immune responses by polarizing T-helper (Th) cells. In allergic disease this response is dominated by Th2 cells. It is still unclear whether the activation of Th cells by DCs in atopic individuals is allergen specific. Methods: Monocyte-derived DCs (MoDCs) obtained from polysensitized patients were stimulated with purified Bet v 1, Phl p 5 and Act d 10, and the surface marker expression was analysed. Proliferation and cytokine profiles of autologous naïve CD4+ T cells co-cultured with allergen-pulsed MoDCs were assessed. Results: The addition of either Bet v 1 or Phl p 5 did not further increase the expression of surface markers from matured MoDCs in all study groups. In co-cultures, autologous naïve CD4+ T cells proliferated when DCs obtained from individuals allergic to birch and grass pollen were stimulated with Bet v 1 and Phl p 5, respectively. In the co-culture supernatants, significantly increased levels of IL-5 and IL-13 were detected. This effect correlated with the sensitization background and was absent when applying an unspecific allergen, Act d 10. The levels of IL-10 in supernatants of MoDCs and the levels of IL-10 and IFN-γ in supernatants of T cells remained unchanged upon stimulation with allergens. Conclusions: In this study we observed that allergen-specific stimulation of MoDCs induces T-cell proliferation and upregulation of Th2-type cytokines. Interestingly, this Th2 polarization was only observed in cells stimulated with the allergen to which the patients were sensitized.

Effects of micro environmental factors on natural killer activity (NK) of Beta Thalassemia major patients

The physiological mechanisms of decreased NK activity of β-Thalassemia major (BTM) patients are unknown. To assess in vitro effects of mononuclear cells and their cytokine secretion on NK activity, we compared activator receptor levels and cytotoxic activity of purified NK cells and NK cells in mononuclear cells (MNC) pools. We collected cell supernatant from unincubated and incubated MNC with K562 cells and measured their secreted cytokines levels. CD16 was lower on the surface of NK cells in MNC pools from BTM patients compared to healthy volunteers. This inhibition does not appear when NK cells were purified. NKp30 levels in NK cells decreased both as purified cells and as part of a pool of MNC in BTM patients. After incubation of MNC pools with K562 target cells, we found that supernatant levels of IL10, TGFβ1 and IL15 cytokines were also significantly higher in BTM patients compared to healthy volunteers.

Immunomodulatory effect of R848 on cytokine production associated with Schistosoma mansoni infection

This work aimed to determine the effect of resiquimod (R848), a low molecular weight imidazoquinolinamine compound with properties as immune response modifiers, on cytokine production in patients infected with Schistosoma mansoni with various degrees of hepatic fibrosis. Schistosoma-infected patients were classified into four groups according to the degree of hepatic fibrosis as examined by ultrasonography group 0 (no fibrosis), group I (incipient fibrosis), group II (moderate fibrosis), and group III (severe fibrosis). Levels of interferon-γ (IFN-γ), interleukin 4 (IL-4), IL-10, and IL-13 were measured by ELISA in the supernatants of cultured peripheral blood mononuclear cells (PBMC) in response to schistosoma egg soluble antigen (SEA) in the presence and absence of R848. R848 induced a significantly higher level of IL-10 in supernatants of SEA-stimulated PBMC of patients in all groups of hepatic fibrosis except group III. Moreover, we significantly found higher levels of IFN-γ in group 0 and group I and significantly lower level of IL-4 in group 0 after R848 stimulation. However, R848 had no effect on IL-13. Taken together, these results suggest that R848 might be used as an immunopotentiating agent in view of novel therapy or vaccine for S. mansoni infection.

Effects of adenovirus-mediated interleukin-10 gene transfer on apoptosis and insulin secretion function of beta cell

To evaluate the efficacy of adenovirus vector-mediated murine interleukin-10 (mIL-10) gene transfer to rat beta cell-RINm5F cells in vitro and to explore the potential value of gene therapy in type 1 diabetes mellitus. The recombinant adenovirus vector Ad-mIL-10 was constructed and transfected into RINm5F cells (Ad-mIL-10 group). Untransfected RINm5F cells and Ad-eGFP-transfected cells were used as controls. The expression of mIL-10 was examined by RT-PCR and Western blot. The levels of IL-10 in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). For a determination of insulin release, the cells were cultured with high glucose (16.7 mmol/L) for a 1-hour co-incubation. Then radioimmunoassay was used to detect the level of insulin in supernatant. After induction of IL-1beta, the levels of nitric oxide (NO) and nitric oxide synthase (NOS) were measured, the apoptosis of transfected cells was detected by Hoechst 33258 staining and Fas expression by flow cytometry. Both mRNA and protein of Ad-mIL-10 were successfully expressed in RINm5F cells. Expression of mIL-10 gene resulted in significant increases in insulin secretion in response to high glucose. Compared with uninfected control and Ad-eGFP infected group, Ad-mIL-10 infected group had decreased levels of NO and NOS induced by IL-1beta [NO level (nmol/10(6) cells): 52.9 +/- 3.2 vs. 227.3 +/- 26.4, 235.1 +/- 28.6, both P < 0.05; NOS level (U/10(6) cells): 9.3 +/- 1.2 vs. 29.8 +/- 2.5, 30.5 +/- 2.8, both P < 0.05]. Furthermore, Ad-mIL-10 gene transfer led to a profound reduction of Fas-expressing islet cells under the induction of IL-1beta. Fas-expressing islet cells in Ad-mIL-10 group were significantly lower than those in uninfected group and Ad-eGFP-transfected group (24.6% +/- 1.0% vs. 33.3% +/- 5.1%, 32.6% +/- 1.1%) (P < 0.05). The apoptotic rates of Ad-mIL-10 group were lower in comparison with the other two groups (9.4% +/- 1.1% vs. 19.2% +/- 2.2%, 20.6% +/- 2.3%, P < 0.05). IL-10 gene transfer to islet beta cells may be beneficial in maintaining beta cells function, protecting islet cells from IL-1beta-mediated apoptosis and promoting islet cells survival. It is a potential therapy for type 1 diabetes mellitus.

Immunogenicity of a Whole-Cell Pertussis Vaccine with Low Lipopolysaccharide Content in Infants

The lack of a clear correlation between the levels of antibody to pertussis antigens and protection against disease lends credence to the possibility that cell-mediated immunity provides primary protection against disease. This phase I comparative trial had the aim of comparing the in vitro cellular immune response and anti-pertussis toxin (anti-PT) immunoglobulin G (IgG) titers induced by a cellular pertussis vaccine with low lipopolysaccharide (LPS) content (wP(low) vaccine) with those induced by the conventional whole-cell pertussis (wP) vaccine. A total of 234 infants were vaccinated at 2, 4, and 6 months with the conventional wP vaccine or the wP(low) vaccine. Proliferation of CD3(+) T cells was evaluated by flow cytometry after 6 days of peripheral blood mononuclear cell culture with stimulation with heat-killed Bordetella pertussis or phytohemagglutinin (PHA). CD3(+), CD4(+), CD8(+), and T-cell receptor gammadelta-positive (gammadelta(+)) cells were identified in the gate of blast lymphocytes. Gamma interferon, tumor necrosis factor alpha, interleukin-4 (IL-4), and IL-10 levels in supernatants and serum anti-PT IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). The net percentage of CD3(+) blasts in cultures with B. pertussis in the group vaccinated with wP was higher than that in the group vaccinated with the wP(low) vaccine (medians of 6.2% for the wP vaccine and 3.9% for the wP(low) vaccine; P = 0.029). The frequencies of proliferating CD4(+), CD8(+), and gammadelta(+) cells, cytokine concentrations in supernatants, and the geometric mean titers of anti-PT IgG were similar for the two vaccination groups. There was a significant difference between the T-cell subpopulations for B. pertussis and PHA cultures, with a higher percentage of gammadelta(+) cells in the B. pertussis cultures (P < 0.001). The overall data did suggest that wP vaccination resulted in modestly better specific CD3(+) cell proliferation, and gammadelta(+) cell expansions were similar with the two vaccines.

IL-10 Production Is Reduced by Hypothermia but Augmented by Hyperthermia in Rat Microglia

Pro-inflammatory cytokines and nitric oxide (NO) are considered responsible for exacerbating brain injury. Activated microglia produce these potentially cytotoxic factors during neuron destruction. The beneficial effects of hypothermia on neuroprotection are considered to be due, in part, to suppression of post-injury inflammatory factors by microglia. However, the underlying mechanisms remain unclear. In particular, the hypothermia's role in modulating anti-inflammatory cytokines is unknown. We examined whether altering culture temperature modifies microglial production of cytokines and NO. Microglia isolated from neonatal rats were cultured with 1 microg/mL lipopolysaccharide (LPS) under hypothermic, normothermic, and hyperthermic conditions for 72 h. Interleukin (IL)-6 and IL-10 levels in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). NO production was analyzed by colorimetric assay of nitrite accumulated in the medium. Compared to normothermia, hypothermia decreased LPS-induced IL-6 production at 6 h of culture. In contrast, hyperthermia reduced IL-6 production throughout culture. IL-10 production was reduced by hypothermia but augmented by hyperthermia at 24-72 h. NO production was reduced by hypothermia throughout culture, while no significant differences in NO production were observed between normothermia and hyperthermia. In this study, hypothermia reduced production of IL-6, IL-10, and NO by LPS-activated microglia, suggesting that the neuroprotective effects of hypothermia might involve not only the inhibition of inflammatory factors, but also anti-inflammatory factor(s). Hyperthermia specifically increased IL-10 production in these cells. These temperature-dependent changes in IL-10 production may imply an important clinical marker for this cytokine in hypothermia-related neuronal protection and in hyperthermia-related neuronal injury.

Modulation of Allergy Incidence in Icelandic Horses Is Associated with a Change in IL-4-Producing T Cells

Background: Equine insect bite hypersensitivity (IBH) is an immediate-type hypersensitivity reaction provoked by insect-derived allergens. Icelandic horses living in Iceland do not have IBH due to absence of relevant insects, but acquire it at high frequency after being imported to mainland Europe. In contrast, their offspring born in mainland Europe has reduced IBH incidence. T helper 1 (Th1) and Th2 cells and cytokines were determined in Icelandic horses born in Iceland and on the continent and which either have IBH or are healthy. Methods: Peripheral blood mononuclear cells (PBMC) from these horses were stimulated for 18 h dur- ing summer and winter with polyclonal T cell stimuli, IBH allergen(s) or irrelevant allergen(s). Cells were analysed by flow cytometry for interferon-γ (IFN-γ) and interleukin-4 (IL-4); RNA was analysed for IFN-γ, IL-4, IL-5 and IL-13 mRNA. Results: During summer, but not during winter, IBH PBMC stimulated polyclonally showed reduced IFN-γ mRNA and IFN-γ-producing cells when compared with those of healthy horses, regardless of origin. PBMC stimulated polyclonally or with IBH allergen showed increased IL-4 mRNA levels and higher numbers of IL-4-producing cells when born in Iceland or showing IBH symptoms. IL-5 and IL-13 mRNA were modulated neither by disease nor by origin. Abrogation of IL-4 production in healthy horses born in mainland Europe may be due, at least in part, to IL-10. There was an increased level of IL-10 in supernatants from PBMC of healthy horses born in mainland Europe and stimulated polyclonally or with IBH allergen. Conclusions: Modulation of IBH incidence is governed by altered Th1/Th2 ratio, which might be influenced by IL-10.

Effect of Neutrophil Apoptosis on Monocytic Cytokine Response to Porphyromonas gingivalis Lipopolysaccharide

Neutrophil apoptosis may play a critical role in the resolution of inflammation by stimulating anti-inflammatory cytokine generation from monocytes. In this study, we investigated the effect of apoptotic neutrophils on interleukin (IL)-10 and IL-1beta production from monocytes in response to Porphyromonas gingivalis lipopolysaccharide. Peripheral blood neutrophils from healthy individuals were isolated by sodium diatrizoate density gradient centrifugation. In order to induce apoptosis, neutrophils were cultured for 24 hours in modified Dulbecco's medium supplemented with 10% autologous serum. Cell apoptosis was quantified by Annexin V positivity and loss of CD16 expression on the cell surface. Peripheral blood mononuclear cells were isolated from the same subjects; monocytes were purified by magnetic cell sorting and cultured with or without apoptotic or fresh neutrophils. Lipopolysaccharide from Porphyromonas gingivalis was used for cell stimulation. IL-1beta and IL-10 levels in supernatants were determined by enzyme-linked immunosorbent assay (ELISA). IL-10 generation was significantly increased in monocytes cultured with apoptotic neutrophils compared to monocytes alone or cocultured with fresh neutrophils (P <0.05). IL-1beta was suppressed both in resting and lipopolysaccharide-stimulated monocytes in the presence of apoptotic neutrophils compared to monocytes alone or monocytes cultured with fresh neutrophils at all time points (P <0.05). Neutrophil apoptosis provides a signal to monocytes, changing the phenotype of the monocyte resulting in the production of anti-inflammatory cytokines and suppression of proinflammatory cytokines in response to lipopolysaccharide.

Inflammatory cytokines and cytokine antagonists in whole blood cultures of patients with systemic juvenile chronic arthritis.

In the present study, we investigated the kinetics and the activation thresholds for the production of a number of pro-inflammatory cytokines and cytokine antagonists in Escherichia coli lipopolysaccharide (LPS) or phytohaemagglutinin (PHA) stimulated whole blood cultures of 13 patients with systemic juvenile chronic arthritis (SJCA) and 10 healthy children. In unstimulated cultures, the levels of interleukin (IL)-1beta, IL-6 and tumour necrosis factor alpha (TNF-alpha) were undetectable in both groups, suggesting that there was no spontaneous production of these cytokines by circulating leucocytes. The activation thresholds for the production of these cytokines, as well as the capacity for production, did not differ significantly between patients and controls. The level of interleukin-1 receptor antagonist (IL-1ra) in plasma of the patients was significantly elevated, while the in vitro production of IL-1ra was essentially normal and it did not correlate with plasma levels of IL-1ra. Supernatant levels of soluble TNF-alpha receptor (sTNF-R) I and II were both significantly elevated and correlated with the global activity score. In contrast, the supernatant levels of IL-10 were reduced in both PHA- and LPS-driven cultures. Although IL-10 levels did not correlate with laboratory or clinical indices of disease activity, the results suggest that reduced IL-10 production may play a pathogenetic role in SJCA.