Down-modulation of mycobacterial-induced IL-1β production in human mononuclear cells by IL-4

Tuberculosis is characterized by a cellular immune response mediated by various cytokines, including IL-1β released by stimulated mononuclear cells. It is now well established that IL-1β plays an important role in local and systemic inflammatory response in tuberculosis. Here we have demonstrated, for the first time, that addition of IL-4 to human mononuclear cells obtained from 11 healthy bacille Calmette–Guérin (BCG)-vaccinated donors reduced BCG-induced production of IL-1β by 91.46 ± 2.2%. This inhibitory effect was found highly significant (P < 0.001) and was dose-dependent. The effect of IL-4 on the secretion of IL-1β was specific, since a complete reversion was obtained with a neutralizing MoAb to human IL-4. In addition, this inhibitory effect was not attributed to a cytotoxic effect, since trypan blue exclusion studies indicated no loss of cell viability in response to IL-4. Interestingly, the induction of IL-1β was regulated by IL-4, at least in part, by a direct mechanism mediated through the 130 extracellular domain of the IL-4 receptor, as demonstrated by incubation of the mononuclear cells with the neutralizing anti-IL-4 receptor MoAb. Finally, a significant down-regulation of IL-1β secretion was observed in hsp70-stimulated mononuclear cells cultured with IL-4. Further experimental work is needed to establish the relevance of IL-4 in human mycobacterial infection in vivo. However, an understanding of the mechanisms that control IL-1β secretion in human mycobacterial infections is essential to understand the pathogenesis of tuberculosis.     

In vitro inhibition of IL-2 biosynthesis in activated human peripheral blood mononuclear cells by a trauma-induced glycopeptide

Traumatic injury often results in profound immunopathology that can lead to immunosuppression, thereby increasing the morbidity and mortality due to sepsis. The isolation and partial characterization of an immunosuppressive glycopeptide (SAP) from serum of severely burned patients has previously been reported by our laboratory. Recently, this trauma peptide has also been identified in the serum of patients with multiple blunt trauma. This glycopeptide is capable of suppressing neutrophil chemotaxis, T-cell blastogenesis and the lysis of human erythrocytes. We demonstrate in this report that SAP inhibits interleukin 2 (IL-2) biosynthesis by mitogen-stimulated peripheral blood mononuclear cells. Peptide concentrations of 50 nmol and above significantly inhibited IL-2 production. Inhibition was not reduced by the addition of indomethacin or anti-PGE2 to cultures containing greater than 100 nmol of peptide, suggesting that inhibition is not entirely prostaglandin-mediated. Preliminary studies have shown that IL-2 suppression by SAP can be partially reversed by the addition of calcium ionophore. These results suggest a potential immunosuppressive mechanism of the trauma peptide in which T cell blastogenesis is inhibited by interference in IL-2 biosynthesis.     

The pneumotoxicant paraquat potentiates IL-1 and TNF production by human mononuclear cells

Interleukin 1 (IL-1) and tumor necrosis factor (TNF) have been implicated in the pathogenesis of bleomycin- and silica-induced lung damage. We have studied the effect of paraquat (PQ), a well-known pneumotoxicant, on IL-1 and TNF production by human peripheral blood mononuclear cells from different healthy donors stimulated with endotoxin. PQ (100 M) potentiated IL-1 production (2–40 fold) and TNF production (2–18 fold). It is, therefore, possible that IL-1 and TNF are also involved in the pneumotoxic action of PQ.     

Secretion of IL-6, IL-8 and G-CSF by human endothelial cells in vitro in response to Staphylococcus aureus and staphylococcal exotoxins

The capacity of endothelial cells to produce and release cytokines (IL-6, IL-8 and G-CSF) in response to exposure to Staphylococcus aureus strains or staphylococcal exotoxins (alpha-toxin, enterotoxin A and TSST-1) was investigated. An endothelial cell culture model of human umbilical vein endothelial cells (HUVEC) was used. Five out of ten clinical isolates of S. aureus were found to induce cytokine production and release from endothelial cells. Four of the five isolates that induce cytokine release produced enterotoxin A, B, C, D and/or TSST-1, compared with two of those that did not induce release. Purified staphylococcal exotoxins (1 pg/ml-1 microg/ml) did not act as primary stimuli and induced no detectable cytokine secretion. When endothelial cells were prestimulated with IL-1beta or TNF alpha at a concentration of 1 ng/ml for 2 h, IL-1beta served as a potent primary stimulus for IL-6, IL-8 and G-CSF production, whereas TNF alpha did not induce any significant cytokine release during the subsequent 24 h. A further increase in IL-6 and G-CSF release, but not of IL-8, was observed when IL-1beta prestimulated cells were exposed to alpha-toxin or TSST-1. However, to potentiate cytokine production (IL-6 and IL-8) by SEA, both IL-1beta and the toxin had to be present simultaneously. Our data show that S. aureus, but not staphylococcal exotoxins, have the capacity to act as primary stimuli of endothelial cells and induce production and release of cytokines. IL-1beta may prime HUVEC to release IL-6, IL-8 and G-CSF prior to subsequent stimulation with staphylococcal exotoxins.     

Modulation of IL-1 alpha, IL-1 beta, and 25K Mr non-IL-1 activity released by human mononuclear cells

To determine if the release of IL-1 alpha and IL-1 beta by cultured PBMC could be independently modulated by different exogenous stimuli, we examined the effect of LPS, IFN gamma, latex beads, and indomethacin on the release of IL-1 alpha and IL-1 beta. PBMC culture supernatants were fractionated by Sephacryl-S-200 column chromatography or HPLC (TSK G3000SW), and each fraction was tested for thymocyte mitogenic activity in the presence or absence of preincubation with anti-IL-1 alpha or anti IL-1 beta monoclonal antibody (mAb) and for the presence of IL-1 alpha or IL-1 beta protein by ELISA. In all experiments, thymocyte mitogenic activity not neutralizable by anti-IL-1 alpha or anti-IL-1 beta mAb was detected in the 25K Mr range, which ranged from 12 to 50% of the total thymocyte mitogenic activity released, depending on the stimuli. Cultured PBMC from 95% of individuals release thymocyte mitogenic activity in the absence of exogenous stimuli, which was increased 1.3-to 7-fold by lopopolysaccharide (LPS) (25-50 micrograms/ml). All of this increased activity was due to increased release of IL-1 beta and non-IL-1 thymocyte mitogenic activity, with no change in the total amount of IL-1 alpha released. Indomethacin (0.1 microgram/ml) induced release of increased thymocyte mitogenic activity of 1.3- to 1.4-fold over unstimulated cultures. All of this increased activity was due to increased release of IL-1 alpha and non-IL-1 activity with a concomitant decrease in IL-1 beta release. Interferon gamma (40-100 U/ml) increased the amount of IL-1 alpha and decreased IL-1 beta and non-IL-1 activity released, resulting in no overall change in the total amount of thymocyte mitogenic activity. Molecular weight fractionation of the PBMC culture supernatants revealed that thymocyte mitogenic activity eluting in the 25K Mr range was not due to IL-1 alpha or IL-1 beta. With certain culture conditions, thymocyte mitogenic activity was detected in the 30-40K Mr range. PBMC cultured with LPS and latex beads in the absence of serum released 30-40K Mr IL-1 alpha, as well as 17K Mr IL-1 alpha and 17K Mr IL-1 beta. PBMC cultured in 2% fetal calf serum (FCS) alone from some donors released only 30-40K Mr thymocyte mitogenic activity. Both IL-1 alpha and IL-1 beta protein was detected by ELISA in this Mr range but only the IL-1 alpha was bioactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-12 inhibits in vitro immunoglobulin production by human lupus peripheral blood mononuclear cells (PBMC)

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by polyclonal B cell activation and by the production of anti-double-stranded (ds) DNA antibodies. Given the inhibitory effects of IL-12 on humoral immune responses, we investigated whether IL-12 displayed such an activity on in vitro immunoglobulin production by SLE PBMC. Spontaneous IgG, IgG1, IgG2, IgG3 and IgM antibody production was dramatically reduced by addition of IL-12. These results were confirmed by Elispot assays detecting IgG- and anti-dsDNA-secreting cells. While IL-6 and TNF titres measured in PBMC supernatants were not modified by addition of IL-12, interferon-gamma (IFN-γ) titres were up-regulated and IL-10 production down-regulated. Since addition of IFN-γ did not down-regulate immunoglobulin production and since the inhibitory activity of IL-12 on immunoglobulin synthesis was not suppressed by anti-IFN-γ antibody, we concluded that the effect of IL-12 on immunoglobulin production was not mediated through IFN-γ. Our data also argue against the possibility that down-regulation of endogenous IL-10 production was responsible for the effect of IL-12. Thus, inhibition of IL-10 production by IFN-γ was not accompanied by inhibition of immunoglobulin production, and conversely, restoration of IL-10 production by anti-IFN-γ antibody did not suppress the inhibitory activity exerted by IL-12 on immunoglobulin production. Taken together, our data indicate that reduction of excessive immunoglobulin and anti-dsDNA antibody production by lupus PBMC can be achieved in vitro by IL-12, independently of IFN-γ and IL-10 modulation.     

In vitro effects of gold sodium thiomalate on IL-1, IL-2 production, IL-2 receptor expression and IL-2 responsiveness in thymocytes and peripheral blood mononuclear cells.

The aim of this study was to examine the effect in vitro of gold sodium thiomalate (GST) on interleukin 1 (IL-1), and interleukin 2 (IL-2) production and IL-2 receptor expression in thymocytes of mice and in human peripheral blood mononuclear cells (PBMC). GST increased the proliferation of thymocytes and PBMC to suboptimal doses of T cell mitogens Con A and PHA. It induced IL-1 production of the accessory adherent cells within thymocytes and PBMC, IL-1 production in the murine macrophage line P388D1 and induced the PHA reactive thymocytes to produce IL-2 and to express IL-2 receptors. The significance of these findings is discussed.     

A multidonor ELISPOT study of IL-1 beta,IL-2, IL-4, IL-6, IL-13, IFN-gamma and TNF-alpha release by cryopreserved human peripheral blood mononuclear cells

Utilization of cryopreserved peripheral blood mononuclear cells (PBMCs), rather than fresh ones collected from the same donor on different dates, overcomes the variability in sensitivity of these cells to activation agents. To understand the effect of cryopreservation, frozen PBMCs from eight healthy donors were studied to release T(H)1 or T(H)2 cytokines including IL- 1 beta, IL-2, IL-4, IL-6, IL-13, TNF-alpha and IFN-gamma using ELISPOT assay. The number of spot-forming cells (SFC) was determined using three concentrations of PBMCs (5 x 10(6), 5 x 10(5) and 5 x 10(4) cells/ml). PBMCs from all eight donors were found to retain their functional capacity to release T(H)1 or T(H)2 cytokines after freezing and thawing. When PBMCs were taken in concentrations 5 x 10(6) or 5 x 10(5) cells/ml, the density of IL-1 beta-, IL-2-, IL-6- and TNF-alpha-related spots in a well for most of the donors appeared to be overly high, making SFC quantification either difficult or impossible. To the contrary, PBMCs in concentration 5 x 10(4) produced distinct and quantifiable spots. The density of spots related to IL-4 and IL-13 release appeared to be optimal for SFC quantification when PBMCs were taken in concentration 5 x 10(6) whereas in 5 x 10(5) cells/ml the spot density was very low and absent in 5 x 10(4) cells/ml concentration group. No relationship between release levels of different cytokines was found, except IFN-gamma and IL-2 cytokine indicating that cryopreserved PBMCs with a high IFN-gamma response will likely have a high IL-2 response as well. Our results indicate that a release level of one cytokine may not be reliably predicted by knowing the level of the other. This implies that it is necessary to test cryopreserved PBMCs in a broad range of concentrations to determine one, which will be optimal for producing distinct and quantifiable spots.     

Production of IL-1beta, IL-1 receptor antagonist and IL-10 by mononuclear cells from patients with SLE.

Depositions of immune-complexes are responsible for many of the pathological features of systemic lupus erythematosus (SLE). For example, immune-complex-induced tissue damage in glomerulonephritis has been shown to be mediated, at least in part, by interleukin (IL)-1. Inappropriate production or function of IL-1 may therefore contribute to disease manifestations in SLE. We investigated lipopolysaccharide (LPS)- and adherent IgG-stimulated release of IL-1beta, IL-1 receptor antagonist (IL-1ra) and IL-10, a potent modulator of IL-1, by blood mononuclear cells from patients with SLE. Mediator production was measured as ng cytokines/10(6) monocytes and compared with clinical parameters. Release of IL-1beta was only detectable in LPS-stimulated cultures and substantially reduced in patients with both active and inactive disease (P < 0.001). LPS-stimulated IL-1ra release was normal and the IL-1ra/IL-1beta ratio was therefore increased (P < 0.05) and correlated inversely to prednisolone dosage (P = 0.009). IgG-stimulated release of IL-1ra was reduced in patients with active disease compared to those with inactive disease and controls (P = 0.002). IL-10 release was similar in patients and controls. We conclude that monocytes from patients with active SLE are deficient in Fc gamma-R-mediated production of IL-1ra, whereas LPS-stimulated IL-1beta release by SLE monocytes is reduced regardless of disease activity. The former may contribute to immune-complex-mediated tissue damage in SLE.     

Eosinophil locomotion and the release of IL-3 and IL-5 by allergen-stimulated mononuclear cells are effectively downregulated in vitro by budesonide

Background Treatment of aliergic asthma with inhaled corticosteroids, such as budesonide (BDN), results in downregulation of T-cell activation and of eosinophil recruitment. Objective Since blood concentrations of BDN, although significantly lower than those measured in the lung, may still have anti-inflammatory effects, we evaluated the activity of BDN in vitro on: allergen-induced release of lymphokines involved in eosinophil chemotaxis (i.e. IL-3 and IL-5), at drug concentrations similar to those obtained in vivo in the lung (10^?8 M), and eosinophil locomotion, at ‘systemic concentrations’ of the drug (10^?10 M and 10^?9M). Methods Twenty-three atopic asthmatic subjects (atopics) sensitized to Dermatophagoides pteronyssinus (Dp) and seven non-atopic healthy subjects (controls) were studied. Purified blood mononuclear cells (BMC) were stimulated with Dp, with or without BDN 10^?8 M and, after 6 days, the supernatants were collected and frozen to test their ehemotactie activity toward purified blood eosinophils and their levels of interleukin (IL)-3 and IL-5 by immunoassay. BMC were then pulsed for additional 18h with [³H]thymidine to evaluate allergen-induced T-cell proliferation. In addition, to test possible direct effects of ‘systemic concentrations’ of the drug on eosinophil locomotion, blood eosinophils were incubated for 1 h with BDN (10^?10 M and 10^?9 M) prior to test their ehemotactie response toward recombinant human IL-3 and IL-5. Results Stimulation of BMC from atopies with Dp induced a statistically significant increase in [H]thymidine incorporation (P < 0.05); secretion of ehemotactie factors for eosinophils (P < 0.001) and the release of IL-3 and IL-5 (P < 0.005 and P < 0.05 respectively). BDN, at the concentration of 10^?8 M, was able to significantly down-regulate T-cell proliferation (P < 0.05), the secretion of ehemotactie factors for eosinophils (P < 0.001) and the release of IL-3 and IL-5 (P < 0.01 and P < 0.05 respectively). Similarly, “systemic concentrations” of BDN (10^?10 M and 10^?9 M) totally inhibited the ehemotactie response of blood eosinophils toward recombinant human IL-3 and IL-5 (P < 0.005). Conclusions Concentrations of BDN similar to those obtained in vivo are effective in inhibiting both the release of eosinophils chemotaxins by allergen-activated mononuclear cells and eosinophil locomotion.     

Functional antagonism between IL-2 and PGA1 or PGJ2 in the control of proliferation of human cord blood-derived mononuclear cells

Cyclopentenone prostaglandins PGA₁ and PGJ₂ induce growth arrest at the G₁/S interphase of the cell cycle in tumour cell lines. Notably, PGE, the precursor molecule of PGA, downregulates the interleukin (IL)-2-dependent proliferation of lymphocytes. Therefore the IL-2/IL-2 receptor system and relative signal transduction is a possible target of the antiproliferative effect of PGA/PGJ. In the present study the PGA₁/PGJ₂-dependent growth inhibition of IL-2-stimulated primary human cord blood mononuclear cells (CBMCs) was found to be mediated by interference with the IL-2 proliferative signal. Both prostaglandins (PGs) inhibited the synthesis of total RNA and protein in IL-2 stimulated cells. PGA₁ and even more PGJ₂ downregulated the expression of IL-2 receptor α (CD25 phenotype). IL-2 partly reversed this effect. Moreover, suppression of IL-2-stimulated cells was not the result of PG-mediated activation of apoptosis. On the contrary, PGs reduced both apoptosis and the high expression of c-Jun detectable in CBMCs spontaneously. Cyclin A/Cdk2 complexes regulate G₁/S transition during the cell cycle. In IL-2-stimulated cells, the levels of Cdk2 were found to be lower in PG-treated cells than those detected in controls. In conclusion, cyclopentenone PGs inhibit CBMCs spontaneous or IL-2-dependent proliferation in part by interfering with the I L-2 pathway.     

In vitro production of IgE-binding factors by human mononuclear cells.

This study documents the production of IgE-binding factors (IgE-BFs) by unstimulated and by mitogen-activated human mononuclear cells. IgE-BFs were detected by a sensitive radioimmunoassay employing monoclonal antibodies to lymphocyte Fc epsilon R (MabER). IgE-BFs were found in the 24-hr CSN of unfractionated tonsillar lymphocytes and of their B-cell but not of their T-cell enriched fractions. When cultured for 1 week, PBMC spontaneously synthesized and released IgE-BFs in the CSN; this was significantly reduced by IgE (10 micrograms/ml). PWM, PHA and Con A significantly increased the production of IgE-BFs by PBMC, and this was not influenced by IgE. The production of IgE-BFs in response to mitogens required interactions between T and non-T cells, and IgE-BFs seemed to be derived mainly from non-T cells. However, low levels of IgE-BFs could be detected in the CSN of highly purified T cells cultured for 1 week in the presence of PHA. The production of IgE-BFs by non-T cells was T-cell dependent and it was mediated by soluble factors released from mitogen-activated T cells. T-cell factors increased the secretion of IgE-BFs by: the macrophage cell line U937, adherent cells, and adherent cell-depleted B-cell preparations. It is concluded that the majority of IgE-BFs produced by cultured human mononuclear cells are derived from B cells and monocytes, and that their production is regulated by T lymphocytes.     

Inhibition of varicella-zoster virus in vitro by human peripheral blood mononuclear cells.

A functional in vitro assay of cell-mediated immunity to varicella-zoster virus (VZV) is described. This procedure uses an enzyme-linked immunosorbent assay (ELISA) to measure the inhibitory effect of human peripheral blood mononuclear cells on VZV antigen production by VZV-infected cell monolayers. When mononuclear cells from VZV-immune, tetanus-immune donors were stimulated with either VZV antigen or tetanus toxoid they reduced VZV antigen production. In contrast, mononuclear cells from VZV-nonimmune, tetanus-immune donors reduced VZV antigen only when stimulated with tetanus toxoid, but not when stimulated with VZV antigen. Cell-free supernatants recovered from the VZV inhibition assays contained the anti-VZV activity. The magnitude of the anti-VZV activity of the supernatants equalled the inhibition observed when the stimulated mononuclear cells were added to the VZV-infected monolayers. Treatment of either mononuclear cells or supernatants with anti-interferon gamma antibody indicated that their VZV inhibitory capability was largely due to the production of interferon gamma by stimulated mononuclear cells.     

Concentrations of immunoreactive human tumor necrosis factor alpha produced by human mononuclear cells in vitro

The concentrations of tumor necrosis factor (TNF) produced by human peripheral blood mononuclear cells (MNC) were measured using a radioimmunoassay (RIA) for human TNF. This was developed using a rabbit antiserum against human recombinant TNF (Hu rTNF), and Hu rTNF labeled with Na125I by a modification of the chloramine T method. This RIA does not detect human lymphotoxin, interleukin-1 alpha or beta, interleukin 2, interleukin 6, interferon alpha or gamma, granulocyte-macrophage-colony stimulating factor, and C5a des arg. A good correlation (r = 0.89) was found between the RIA and the cytolytic bioassay for TNF. The sensitivity of the RIA is between 3 and 78 pg/ml (median 11 pg/ml). The mean concentration of TNF in 24-h culture supernatants of human MNC exposed to different concentrations of lipopolysaccharide (LPS) was found to increase in dose-dependent fashion and then level off between 50 and 100 ng/ml. The concentrations of IL-1 beta and alpha detected by specific RIAs in these supernatants were between 0.2 and 19 ng/ml and 0.04 and 1 ng/ml, respectively. The amount of TNF produced by human MNC in vitro was determined in a cohort of 50 normal volunteers. Without exogenous stimuli, TNF concentrations were almost always below the detection limit; with 0.5 ng/ml LPS, the median concentration of TNF was 2 ng/ml, and with PHA the median was 3.8 ng/ml. In cultures performed in the presence of indomethacin significantly (p less than 0.005) more TNF was produced. Using this RIA, we could detect TNF in the circulation of mice injected with Hu rTNF. When plasma samples of patients with febrile illnesses were added directly to the RIA, TNF was not detectable, with the exception of patients with malaria. These studies demonstrate the range and sensitivity of LPS-induced and mitogen-induced production of immunoreactive TNF by human MNC in vitro without interference of similar cytokines in bioassays.     

Seasonal variation of IgE synthesis in vitro by human peripheral blood mononuclear cells

Seasonal variations in IgE antibody synthesis in vitro were studied in cultures of blood mononuclear cells (MNC) from 11 pollen allergic individuals. The IgE levels were significantly higher in two summer seasons than in the winter and spring between them. Net synthesis was confined to the summer in all but one of the patients. All the IgE in the cultures outside the pollen season represented preformed IgE which was present mainly (59%) in the monocyte fraction. Thus, preformed IgE seems to persist in monocytes at times when there is little de novo synthesis of IgE.     

IL-6预处理保护H2O2致心肌细胞损伤作用机制初探

目的初步探讨IL-6预处理对H2O2致心肌细胞氧化应激损伤的作用机制。方法采用心肌细胞原代培养方法,以H2O2刺激心肌细胞,建立细胞氧化应激模型;采用MTT法检测细胞活力;AnnexinV-FITC染色法和流式细胞术检测细胞凋亡率;检测心肌细胞内谷胱甘肽（GSH）、超氧化物歧化酶（SOD）、丙二醛（MDA）的表达情况。结果 H2O2可降低心肌细胞存活率并能增加其凋亡,IL-6预处理后能显著改善细胞活力及凋亡情况,与模型组相比差异有统计学意义（P〈0.05）。低浓度IL-6能明显增加细胞内GSH与SOD的水平,并降低MDA的含量,随着IL-6浓度的增加,这种效应逐渐消失。结论 IL-6预处理能保护H2O2致心肌细胞损伤作用,这可能与IL-6调节细胞GSH、SOD、MDA表达有关。     

Methimazole increases H2O2 toxicity in human thyroid epithelial cells

Hydrogen peroxide (H₂O₂) is necessary for thyroid hormone production and also for intracellular signalling purposes. Owing to its oxidative properties, however, it is harmful to cells, and deregulation of thyroid oxidative state has been implicated in the pathology of thyroid cancer. In this project, we studied the effects of H₂O₂ on morphology and histochemical indicators of differentiated function (intracellular thyroglobulin), ability to generate NADPH (glucose-6-phosphate dehydrogenase (G6PD) activity) and vitality (apoptosis assay) in human thyroid epithelial cells. We further evaluated whether methimazole, an antithyroid drug reported to have antioxidative properties, could counteract the effects of H₂O₂. Our data demonstrate tolerance to H₂O₂ in concentrations less than 0.3 mM and harmful effects at higher concentrations. 10 mM methimazole sensitizes the cells towards H₂O₂, possibly due to a dose-dependent inhibition of G6PD. Our data demonstrate the importance of this antioxidative system and point towards a relevant, but seldom recognized, influence of methimazole.