Interleukin-15 augments superoxide production and microbicidal activity of human monocytes against Candida albicans

Overexpression of EGFR and c-erbB2 frequently occurs in human breast cancers, correlating with poor prognosis. Here we show that overexpression of EGFR and c-erbB2 in cell lines increases cell migration, an important step in metastasis formation. The effect of EGFR on migration is dependent on the addition of EGF to the cells. In contrast, c-erbB2 seems to act independently of its ligand in these assays. Overexpression of this receptor is sufficient to induce cell migration. In addition, we investigated the involvement of a number of signal transduction pathways known to be activated by the EGFR. We found that inactivation of MAPKK results in a decreased migration, while inactivation of PI3K increases migration.     

Interleukin-10 in non-Hodgkin's lymphoma

Interleukin-10 (IL-10) is a pleiotropic cytokine produced by type 2 helper cells (Th2), as well as by monocytes and macrophages, and normal and neoplastic B lymphocytes. It is highly homologous to an open reading frame of Epstein-Barr virus (EBV) called BCRF1, and EBV infection of B-cells up-regulates IL-10. IL-10 production has strong immunosuppressive effects via inhibition of Th1 type cytokines, including interferon gamma and interleukin-2. On B-cells, IL-10 has a potent stimulating effect, inducing proliferation and differentiation. Interestingly, in cell lines derived from B-cell lymphomas, IL-10 production has been found to be up-regulated, and it serves as an autocrine growth factor. In patients with non-Hodgkin's lymphoma (NHL), serum IL-10 levels are significantly increased when compared to normal individuals and NHL patients in remission. The prognostic significance of these increased levels vary according to the assay used. Both human IL-10 and viral IL-10 are increased, and when specific assays for human IL-10 are used, there seems to be no prognostic significance, whereas when the assay cross-reacts with viral IL-10, high levels correlate with poor prognosis. These results suggest that viral IL-10 might have some pathogenic role in NHL.     

Interleukin-10 inhibits activation of coagulation and fibrinolysis during human endotoxemia

Interleukin-10 (IL-10) has been found to inhibit lipopolysaccharide (LPS)-induced tissue factor expression by monocytes in vitro. To determine the effects of IL-10 on LPS-induced activation of the hemostatic mechanisms in vivo, we performed a placebo-controlled, cross-over study of human endotoxemia. Two groups of eight volunteers were challenged with LPS (4 ng/kg) on two occasions: once in conjunction with placebo, and once with recombinant human IL-10 (rhIL-10; 25 microg/kg). In group 1, placebo or rhIL-10 was given 2 minutes before LPS challenge, group 2 received placebo or rhIL-10 1 hour after LPS administration. Pretreatment with rhIL-10 reduced both LPS-induced activation of the fibrinolytic system (plasma concentrations of tissue type plasminogen activator, plasmin-alpha2-antiplasmin complexes, and D-dimer), and inhibition of fibrinolysis (plasma levels of plasminogen activator inhibitor 1), whereas posttreatment only inhibited the latter response. Both IL-10 pre- and posttreatment attenuated activation of the coagulation system (plasma levels of prothrombin fragment F1 + 2 and thrombin-antithrombin complexes). These results indicate that rhIL-10, besides its well-described inhibitory effects on cytokine release, potently modulates the fibrinolytic system and inhibits the coagulant responses during endotoxemia.     

Inhibition of IL-10 expression by IFN-gamma up-regulates transcription of TNF-alpha in human monocytes

Stimulation of human monocytes with LPS induces expression of multiple cytokines, including TNF-alpha, IL-1 beta, IL-6, and IL-10, IL-10 expression is delayed relative to that of TNF-alpha, IL-1 beta, and IL-6. Furthermore, IL-10 feedback inhibits expression of TNF-alpha, IL-1 beta, and IL-6, thus providing an efficient autocrine mechanism for controlling proinflammatory cytokine production in monocytes. The Th1-type lymphokine, IFN-gamma, markedly up-regulates TNF-alpha production in monocytes. However, the precise mechanism by which IFN-gamma mediates this effect is unknown. We examined the effects of IFN-gamma on IL-10 expression in LPS-stimulated monocytes, and the relationship between IL-10 and TNF-alpha production in these cells. LPS stimulation induced rapid, ordered expression of multiple cytokines. Steady-state mRNA levels for TNF-alpha increased rapidly, reached maximal levels by 2 to 3 h poststimulation, and then declined sharply. IL-1 beta and IL-6 mRNA levels also increased markedly following stimulation with LPS, but decreased more slowly than did TNF-alpha. Down-regulation of mRNA for TNF-alpha, IL-1 beta, and IL-6 coincided with a delayed and more gradual increase in IL-10 mRNA levels. Furthermore, neutralization of IL-10 with anti-IL-10 Abs prolonged TNF-alpha mRNA expression, and significantly increased net TNF-alpha production. IFN-gamma suppressed expression of IL-10 mRNA and protein in a dose-dependent manner. Moreover, inhibition of IL-10 production correlated with a marked increase in both the magnitude and duration of TNF-alpha expression. Thus, potentiation of TNF-alpha production by IFN-gamma in monocytes is coupled to inhibition of endogenous IL-10 expression.     

Interleukin-4 but not interleukin-10 inhibits the production of leukemia inhibitory factor by rheumatoid synovium and synoviocytes

The expression of the proinflammatory cytokine leukemia inhibitory factor (LIF) has been reported in the cartilage and synovium of rheumatoid arthritis (RA) patients. Here, we show that high levels of LIF were constitutively produced by cultures of synovium pieces. Low levels of LIF were produced spontaneously by isolated synoviocytes, but interleukin (IL)-1 beta caused a fourfold enhancement of this secretion. The anti-inflammatory cytokine IL-4 reduced the production of LIF by synovium pieces by 75%, as observed earlier with IL-6, IL-1 beta and tumor necrosis factor (TNF)-alpha. IL-4 had a direct effect since it inhibited LIF production by unstimulated and IL-1 beta- or TNF-alpha-stimulated synoviocytes. Conversely, IL-4 enhanced the production of IL-6, which shares with LIF biological activities and receptor components. The inhibitory effect of IL-4 was dose dependent and was reversed using a blocking anti-IL-4 receptor antibody. Similar inhibitory action of IL-4 on LIF production was observed on synovium pieces from patients with osteoarthritis and on normal synoviocytes. IL-10, another anti-inflammatory cytokine acting on monocytes, had no effect on LIF production by either synovium pieces or isolated synoviocytes. Thus, the production of LIF by synovium tissue was inhibited by IL-4 through both a direct effect on synoviocytes and an indirect effect by inhibition of the production of LIF-inducing cytokines.     

Interleukin-10 inhibits the in vitro proliferation of human activated leukemic CD5+ B-cells

Umbilical cord blood mononuclear cells isolated by density centrifugation are contaminated by erythrocytes and nucleated erythroid precursors which may exceed 50% of the total cell population, and thus interfere with phenotypic, functional and mRNA analyses. Lysis with hypotonic ammonium chloride can overcome this problem, but interferes with lysosomal function and should be avoided when cell preparations are intended for functional studies. The aim of this study was to develop a technique for removing erythroid cells from cord blood mononuclear cell preparations that would be as effective as ammonium chloride lysis but would not affect cellular function. This paper describes a method using 10F7, a mouse monoclonal antibody against human glycophorin A, and magnetic beads coated with anti-mouse immunoglobulin. The population of cord blood mononuclear cells recovered using this technique was of high purity, good yield and viability, and the cells responded appropriately to stimulation in vitro. To maximise cost-effectiveness, purification with magnetic beads could be performed after two density separations to reduce the quantity of beads required.     

Interleukin-3 inhibits cycloheximide induction of C-jun mRNA in human monocytes: possible role for a serine/threonine phosphatase

Cycloheximide is a strong inducer of the c-jun protooncogene mRNA at concentrations (< or = 50 ng/ml) that do not inhibit protein synthesis in human monocytes. This induction is transient lasting 30-60 min in contrast to the sustained induction obtained with concentrations that inhibit protein synthesis. The pluripotent colony stimulating factor interleukin-3 (IL-3) (10 ng/ml) is also a modest inducer of the c-jun gene in these cells; however, in combination with cycloheximide, IL-3 dramatically reduces the c-jun induction below levels induced by cycloheximide alone. This is a true inhibition and is not due to a change in temporal kinetics of induction because the suppression in the presence of IL-3 is observed at both 30 and 60 min after simultaneous addition of both IL-3 and cycloheximide. Preincubation of monocytes with 12.5 nM okadaic acid (a potent inhibitor of protein phosphatases 1 and 2A) and cycloheximide prior to addition of IL-3 restored the level of c-jun induction to that mediated by cycloheximide alone. This concentration of okadaic acid inhibited almost 70% of the phosphorylase phosphatase activity in monocyte lysates. These observations suggest that activation of protein serine/threonine phosphatase(s) underlies the ability of IL-3 to inhibit cycloheximide induction of c-jun in monocytes.     

Increased production of interleukin-10 by human blood monocytes stimulated with Mycobacterium avium-intracellulare complex

BACKGROUND: Overseas screening of immigrants and refugees applying for a visa to the United States identifies foreign-born individuals who are at high risk for tuberculosis (TB) or who have active TB. The system's effectiveness relies on further medical evaluation and follow-up of foreign-born individuals after their arrival in the United States. METHODS: Retrospective cohort study of 893 immigrants and refugees who arrived in the United States from July 1, 1992, through December 31, 1993, with a destination of San Francisco, Calif, and a referral for further medical evaluation. MAIN OUTCOME MEASURES: Time to report to the local health department after arrival and the yield of active and preventable cases of TB from follow-up medical evaluations. RESULTS: Median time from arrival in the United States to seeking care in San Francisco was 9 days (range, 1-920 days). Of 745 immigrants and refugees (83.4%) who sought further medical evaluation, 51 (6.9%) had active TB and 296 (39.7%) were candidates for preventive therapy. Being a refugee was an independent predictor of failure to seek further medical evaluation in the United States. Class B-1 disease status based on overseas TB screening (odds ratio, 3.5; 95% confidence interval, 2.0-6.2) and being from mainland China (odds ratio, 4.4; 95% confidence interval, 1.9-9.9) were independent predictors of TB diagnosed in San Francisco. CONCLUSIONS: Timely, adequate medical evaluation and follow-up care of immigrants and refugees has a relatively high yield and should be a high priority for TB prevention and control programs.     

Enhancement of macrophage colony-stimulating factor-induced growth and differentiation of human monocytes by interleukin-10

Interleukin-10 (IL-10) has been reported to be a negative cytokine for monocytes/macrophages. In the present study, we showed that IL-10 is rather a positive cytokine and augments the growth and differentiation of human monocytes stimulated with macrophage colony-stimulating factor (M-CSF). Highly purified adherent human monocytes were cultured for 7 days with M-CSF in the presence or absence of IL-10. The number of recovered cells increased in the culture of monocytes with M-CSF + IL-10 compared to the culture with M-CSF alone. IL-10 alone was not enough to maintain the survival and differentiation of monocytes into macrophages. Morphological change cultured in M-CSF was also accelerated by addition of IL-10, and macrophages cultured in M-CSF + IL-10 were more elongated compared to macrophages cultured with M-CSF alone. Binding of 125I-M-CSF to monocytes incubated with M-CSF + IL-10 was about 1.7-fold higher than that to monocytes incubated with M-CSF alone. In accordance with the binding study, Northern blot analysis showed that the levels of the expression of c-fms, M-CSF receptor, mRNA in macrophages cultured in M-CSF + IL-10 were higher than that in macrophages cultured in M-CSF alone. Macrophages cultured in M-CSF + IL-10 expressed higher level of Fc gamma RI, II, III, and showed augmented Fc gamma receptor mediated phagocytosis. The former also produced higher level of H2O2 and O2-, when stimulated with zymosan, and of IL-6 when stimulated with lipopolysaccharide compared to the latter. These results taken together suggest that IL-10 augments the growth and differentiation of human monocytes cultured in M-CSF.     

Interleukin-1 stimulates hypothalamic inhibitory amino acid neurotransmitter release

In order to get a better insight into the function of amino acid residues located in the second transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, all exon 18 mutations found in cystic fibrosis (CF) patients were characterized at the protein and at the electrophysiological level. Of the different mutations present in transmembrane helix 12 (M1137V, M1137R, I11139V and deltaM1140), and the intracytoplasmic loop connecting TM12 and NBD2 (D1152H and D1154G), only M1137R interfered with the proper maturation of the protein. Permeability studies performed after injection of the different wild-type and mutant cRNAs in Xenopus laevis oocytes indicated that the mutations did not alter the permeability sequence of the CFTR channels. The whole cell cAMP activated chloride currents, however, were significantly reduced for M1137V, I1139V, D1152H and D1154G and close to zero for deltaM1140, indicating that these mutations interfere with the proper gating of the chloride channels.     

Induction of interleukin-10 by human immunodeficiency virus type 1 and its gp120 protein in human monocytes/macrophages

In this study, we evaluated the effects of human immunodeficiency virus type 1 (HIV-1) and its gp120 protein on interleukin-10 (IL-10) expression in cultured human monocytes/macrophages. Infection of either 1-day monocytes or 7-day monocyte-derived macrophages with HIV-1 strain Ba-L resulted in clear-cut accumulation of IL-10 mRNA at 4 and 24 h. Likewise, treatment of these cells with recombinant gp120 induced IL-10 mRNA expression and caused a marked increase in IL-10 secretion. Monoclonal antibodies to gp120 strongly inhibited recombinant gp120-induced IL-10 secretion by monocytes/macrophages. Moreover, the addition of IL-10 to monocytes/macrophages resulted in a significant inhibition of HIV-1 replication 7 and 14 days after infection. On the whole, these results indicate that HIV-1 (possibly through its gp120 protein) up-regulates IL-10 expression in monocytes/macrophages. We suggest that in vivo production of IL-10 by HIV-primed monocytes/macrophages can play an important role in the early response to HIV-1 infection.     

Interleukin-10 production by human carcinoma cell lines and its relationship to interleukin-6 expression

Recent data indicate a major role for IL-10 in suppressing immune and inflammatory reactions. To date, expression of human IL-10 has been attributed primarily to helper T lymphocytes, activated monocytes, and neoplastic B cells, and was often found to be associated with IL-6 expression. In this study we sought to determine whether non-hematopoietic human tumor cell lines produce IL-10 and, if so, what is the relationship between IL-10 and IL-6. Using ELISA, we determined IL-10 and IL-6 levels in culture supernatants of 48 cell lines established from carcinomas of the kidney, colon, breast and pancreas, malignant melanomas and neuroblastomas. IL-6 protein was secreted by 28 of the tumor cell lines; IL-10 was measurable in 15 cell lines. IL-6 secretion was maximal and most frequent in renal-cancer cell lines, while IL-10 production was found to be highest and most common among cell lines derived from colon carcinomas. IL-10 in conditioned medium of one of the colon carcinoma cell lines (CCL222) was bio-active, as demonstrated in the mouse MC/9 mast-cell-line assay and in human mixed-lymphocyte reactions. In both assays, IL-10 bio-activity was neutralized by an anti-IL-10 monoclonal antibody. Expression of IL-6 and IL-10 was confirmed by RNA analysis using message amplification by PCR and sequencing of amplified cDNA. LPS, IL-1 alpha, and TNF-alpha strongly enhanced the release of IL-6 by RCC cells, but only marginally affected IL-10 production in colon-carcinoma cells. IL-10 secretion by colon-carcinoma cells was moderately stimulated by IFN-gamma and IL-4. Dexamethasone suppressed the release of IL-6, but had no inhibitory effect on IL-10 secretion. Our results demonstrate that tumor cell lines established from certain types of human carcinomas are capable of expressing and releasing IL-6 and/or IL-10, suggesting a role of these cytokines in solid-tumor development and anti-tumor immunity.     

Borrelia burgdorferi induces chemokines in human monocytes

Lyme disease is clinically and histologically characterized by strong inflammatory reactions that contrast the paucity of spirochetes at lesional sites, indicating that borreliae induce mechanisms that amplify the inflammatory response. To reveal the underlying mechanisms of chemoattraction and activation of responding leukocytes, we investigated the induction of chemokines in human monocytes exposed to Borrelia burgdorferi by a dose-response and kinetic analysis. Lipopolysaccharide (LPS) derived from Escherichia coli was used as a positive control stimulus. The release of the CXC chemokines interleukin-8 (IL-8) and GRO-alpha and the CC chemokines MIP-1alpha, MCP-1, and RANTES was determined by specific enzyme-linked immunosorbent assays, and the corresponding gene expression patterns were determined by Northern blot analysis. The results showed a rapid and strong borrelia-inducible gene expression which was followed by the release of chemokines with peak levels after 12 to 16 h. Spirochetes and LPS were comparably effective in stimulating IL-8, GRO-alpha, MCP-1, and RANTES expression, whereas MIP-1alpha production preceded and exceeded chemokine levels induced by LPS. Unlike other bacteria, the spirochetes themselves did not bear or release factors with intrinsic chemotactic activity for monocytes or neutrophils. Thus, B. burgdorferi appears to be a strong inducer of chemokines which may, by the attraction and activation of phagocytic leukocytes, significantly contribute to inflammation and tissue damage observed in Lyme disease.     

Interleukin-4 and interleukin-10 are chondroprotective and decrease mononuclear cell recruitment in human rheumatoid synovium in vivo

We used the severe combined immunodeficient (SCID) mouse model to assess the effect of interleukin-4 (IL-4) or IL-10 injection on cartilage degradation and mononuclear cell (MNC) recruitment to human rheumatoid synovium in vivo. Human rheumatoid synovium and cartilage from five rheumatoid arthritis patients, obtained after joint replacement surgery, were engrafted subcutaneously to 6-8-week-old SCID CB17 mice. Synovial tissues were injected with recombinant human IL-4 (rhIL-4, 100 ng; rhIL-10, 100 ng), both cytokines, or tumour necrosis factor-alpha (TNF-alpha) (1000 U), or phosphate-buffered saline twice a week for 4 weeks. The graft was removed and immunochemical analysis was carried out to assess intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression. Moreover, cartilage degradation was assessed through the quantification of the erosion surface on a computerized image of the engrafted cartilage at high power view. MNC recruitment in the synovial tissue was determined by labelling blood MNC with indium-111 before their intraperitoneal injection. The activity obtained in the region of the graft were determined with a gamma camera 72 hr postinjection. The results are expressed as a percentage of initial injected activity. After 4 weeks we observed a decrease of cartilage area in controls (77 +/- 8%), inhibited after injection of IL-4, IL-10, or both cytokines (90 +/- 3%, 89.1 +/- 4%, 89.2 +/- 5% respectively), and 57 +/- 17% after TNF-alpha injection. The % MNC activity in the graft decreased to 77 +/- 81% (NS), 9 +/- 4% (P < 0.003) and 19 +/- 6% (P < 0.007) compared with untreated synovial tissue after treatment with IL-4, IL-10, or both cytokines, respectively. Moreover, IL-10 but not IL-4 decreased the expression of ICAM-1 but not VCAM-1 or E-selectin by synovial cells. These results suggest that IL-10 and IL-4 could have chondroprotective properties, and that IL-10 but not IL-4 inhibits MNC traffic towards the synovial tissue efficiently.     

Interleukin-13 inhibits cytokine secretion by blood monocytes from patients with IgA nephropathy

The study of hypertrophic cardiomyopathy has been at the vanguard of molecular genetic investigation into inherited diseases of the cardiovascular system for the greater part of the last decade. These studies have shown it to be a disease of sarcomeric contractile proteins and have highlighted its genetic heterogeneity. There are now seven known genetic loci and six disease genes associated with the condition. The biology of the mutant polypeptides has been studied in vitro, and animal models are being developed. Increased understanding gained from these studies has clarified features of the condition at the clinical level and has had an impact on management of patients. It is hoped that this work will lead to the development of novel therapies for both hypertrophic cardiomyopathy and acquired forms of left ventricular hypertrophy.     

Interleukin-10 inhibits neutrophil phagocytic and bactericidal activity

Effective host defense against bacterial invasion is characterized by the vigorous recruitment and activation of inflammatory cells, which is dependent upon the coordinated expression of both pro- and anti-inflammatory cytokines. Interleukin-10 (IL-10) is a recently described cytokine with potent anti-inflammatory properties in vivo and in vitro. In this study we investigated whether IL-10 could directly regulate the ability of neutrophils (PMN) to phagocytose and kill bacteria. Initial studies demonstrated that human recombinant IL-10 (hrIL-10) inhibited the ability of PMN to phagocytose Escherichia coli in vitro. Inhibition of phagocytosis occurred in the absence of changes in CR1 (C3b) or Fc receptor expression, as treatment of PMN with IL-10 failed to induce significant changes in Fc gamma IIR, Fc gamma IIIR or CR1 cell surface expression. However, incubation of PMN with IL-10 resulted in a dose-dependent decrease in CDIIb (Mac-1) expression. In addition to effects on PMN phagocytosis, hrIL-10 significantly attenuated PMN microbicidal activity, as bactericidal assays revealed that co-incubation of PMN with hrIL-10 resulted in a marked decrease in killing of phagocytosed bacteria. Furthermore, IL-10 inhibited the production of superoxide from PMA-stimulated PMN, suggesting that the detrimental effects of IL-10 on PMN microbicidal activity were due, in part, to suppression of respiratory burst. In summary, our studies indicate that IL-10 inhibits PMN-dependent phagocytosis and killing of E. coli in vitro, and suggest that this cytokine may impair effective antibacterial host defense in vivo.