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F Willems

Publications and source records attributed to F Willems.

At least 37 records · Page 2Linked to original sources
PubMed1999

Interferon-beta inhibits Th1 responses at the dendritic cell level. Relevance to multiple sclerosis.

Clinical studies have demonstrated beneficial effects of interferon-beta (IFN-beta) therapy in multiple sclerosis (MS) patients. However, the mechanism of action of IFN-beta in MS remains unknown. IFN-beta has even been demonstrated to enhance isolated T cell secretion of IFN-gamma, a cytokine proven to be deleterious in MS. However, IFN-beta inhibits IFN-gamma secretion of T cells, when they are stimulated by antigen presenting cells (APC). We therefore decided to study the effects of IFN-beta on the in vitro differentiation of dendritic cells (DC), a major class of APC. First, we found that the addition of IFN-beta at the initiation of the differentiation did not modify DC morphology, but enhanced the expression of molecules involved in antigen presentation (HLA-DR, B7/1 and B7/2). However, DC, differentiated in the presence of IFN-beta, secreted less interleukin-12 (IL-12) both spontaneously and upon activation by CD40-ligand bearing cells. As a consequence, DC differentiated in the presence of IFN-beta induced less IFN-gamma secretion by alloreactive T cells. We conclude that the direct action of IFN-beta on DC results in inhibition of their ability to secrete IL-12 and to elicit Thelper-1 (Th-1) type responses. These results are of particular interest in MS, in which a critical role for IL-12 has recently been suggested by a number of clinical and experimental observations.

Blood Donors↗
PubMed1998

Oxidative stress up-regulates IL-8 and TNF-alpha synthesis by human dendritic cells.

In addition to their damaging effects, reactive oxygen intermediates exert a regulatory role on gene expression and cell apoptosis. In this study, we evaluated the effects of oxidative stress on human dendritic cells (DC), a cell type which is critical for the initiation of the immune response. For this purpose, we tested the effects of H2O2 on DC derived from adherent peripheral blood mononuclear cells cultured in the presence of granulocyte-macrophage colony-stimulating factor and IL-4. Despite a moderate increase of DC apoptosis in the presence of H2O2, we observed that H2O2 stimulated the production of IL-8 and TFN-alpha by DC in a dose-dependent manner. The induction of cytokine synthesis was found to depend on the oxidative properties of H2O2 as it was inhibited by the addition of catalase, and to require de novo protein synthesis as it was not observed in the presence of cycloheximide. These data suggest that DC could contribute to innate immunity through an enhanced production of inflammatory cytokines in response to oxidative stress.

Cell Survival↗
PubMed1997

Thermolabile methylenetetrahydrofolate reductase in coronary artery disease.

BACKGROUND: Hyperhomocysteinemia, an independent and graded risk factor for coronary artery disease (CAD), may result from both environmental and hereditary factors. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of methylenetetrahydrofolate to methyltetrahydrofolate, the methyl donor in the remethylation of homocysteine to methionine. A 677C-->T mutation in the MTHFR gene has been associated with elevated homocysteine concentrations in homozygous (+/+) individuals. METHODS AND RESULTS: We assessed the frequency of this common mutation in 735 CAD patients from the Regression Growth Evaluation Statin Study (REGRESS), a lipid-lowering coronary-regression trial, and in 1250 population-based control subjects. Furthermore, the association between the mutation and serum homocysteine concentrations was studied. The frequency of the homozygous (+/+) mutation was 9.5% among patients versus 8.5% among control subjects, resulting in an odds ratio of 1.21 (95% confidence interval [CI], 0.87 to 1.68), relative to the (-/-) genotype. Homocysteine concentrations were significantly elevated in both (+/+) and (+/-) individuals compared with (-/-) individuals (median homocysteine levels, 15.4, 13.4, and 12.6 micromol/L, for (+/+), (+/-), and (-/-) individuals, respectively). For a summary estimation of the risk of the (+/+) genotype for CAD, we performed a meta-analysis on 8 different case-control studies on thermolabile MTHFR in CAD. In the meta-analysis, the homozygous (+/+) genotype was present in 299 of 2476 patients (12.1%) and in 257 (10.4%) of 2481 control subjects, resulting in a significant odds ratio of 1.22 (95% CI, 1.01 to 1.47) relative to the (-/-) genotype. CONCLUSIONS: Both the homozygous (+/+) and heterozygous (+/-) genotype result in elevated homocysteine concentrations. From our meta-analysis, we conclude that the homozygous (+/+) genotype is a modest but significant risk factor for CAD.

Coronary Disease↗
PubMed1997

Bacterial lipopolysaccharide stimulates the production of cytokines and the expression of costimulatory molecules by human peripheral blood dendritic cells: evidence for a soluble CD14-dependent pathway.

To investigate the responses of dendritic cells (DC) during Gram-negative infections, we analyzed the effects of graded doses of LPS on the cytokine profile, phenotype, and allostimulatory potential of human DC generated by culturing plastic-adherent PBMC in presence of IL-4 and granulocyte-macrophage-CSF. First, we found that LPS stimulates the production of high levels of TNF-alpha, IL-6, IL-8, IL-12 by DC and up-regulates their expression of HLA-DR, B7-1, B7-2, and CD40. The effects of LPS were dose dependent, with a significant stimulatory effect already observed at a concentration of 0.1 ng/ml and a plateau being reached at 10 ng/ml. These phenotypic changes correlated with increased allostimulatory properties of LPS-activated DC because DC treated with LPS were significantly more efficient than untreated DC in eliciting IL-2 and IFN-gamma synthesis by alloreactive T cells and stimulating their proliferation. Experiments using neutralizing anti-IL-12 mAb indicated that LPS-induced IL-12 is responsible for the increased production of IFN-gamma but not for the increased proliferation during MLR. Finally, we observed that the DC responses to low levels of LPS (1 ng/ml) were dramatically inhibited by a blocking anti-CD14 mAb, although DC do not express CD14 molecules on their membrane. Experiments using serum depleted of soluble CD14 (sCD14) and sCD14 either purified from human serum or in recombinant form further established that DC respond to LPS via a soluble CD14-dependent pathway.

Antigens, Surface↗
PubMed1997

Interleukin-10 prevents the generation of dendritic cells from human peripheral blood mononuclear cells cultured with interleukin-4 and granulocyte/macrophage-colony-stimulating factor.

We evaluated the effects of interleukin (IL)-10 on the differentiation of dendritic cells (DC) obtained by culturing plastic-adherent peripheral blood mononuclear cells for 7 days in presence of granulocyte/macrophage-colony-stimulating factor (GM-CSF) + IL-4. The addition of IL-10 at the initiation of culture resulted in the generation of macrophage-like cells with expressing high levels of CD14 and decreased levels of CD1a and CD1c. Furthermore, cells generated in presence of IL-10 secreted lower levels of IL-12, but higher levels of IL-8 compared with DC generated in absence of IL-10, both spontaneously and after CD40 engagement. Finally, cells generated in presence of IL-10 were less efficient than DC in stimulating the production of IL-2, interferon-gamma, and IL-4 by allogeneic T cells. We conclude that IL-10 prevents the generation of DC induced by GM-CSF + IL-4 and favors the development of macrophages with a lower T cell stimulatory potential, but secreting higher levels of IL-8 than DC.

Antigens, CD↗
PubMed1997

Human dendritic cell responses to lipopolysaccharide and CD40 ligation are differentially regulated by interleukin-10.

We evaluated the effects of interleukin (IL)-10 on the maturation of human dendritic cells (DC) induced either by lipopolysaccharide (LPS) or CD40 engagement. For this purpose, DC generated by culturing plastic-adherent peripheral blood mononuclear cells for 7 days with granulocyte/macrophage-colony-stimulating factor and IL-4 were incubated for 3 days with either LPS (10 ng/ml) or 3T6 fibroblasts transfected with the gene encoding CD40 ligand, in absence or presence of IL-10. First we found that the membrane expression of CD83, a marker of mature DC, was inhibited by IL-10 when induced by LPS but not by CD40 engagement. Likewise, IL-10 inhibited LPS-induced but not CD40-dependent CD86 (B7.2) up-regulation on DC. Furthermore, IL-10 inhibited the production of IL-8 and tumor necrosis factor-alpha by DC when activated by LPS but not by CD40. In contrast, IL-10 inhibited IL-12 production in both activation systems. We conclude that IL-10 differentially influences LPS-dependent and CD40-dependent pathways of DC maturation.

Antigens, CD↗
PubMed1997

Cloning of human IL-12 p40 and p35 DNA into the Semliki Forest virus vector: expression of IL-12 in human tumor cells.

IL-12 can enhance the development of effective immune responses against tumors as well as against certain infectious agents. It is therefore a potential candidate for therapeutic use in cancer therapy and in the design of vaccines against several infectious diseases. Several studies have demonstrated that IL-12 could efficiently induce tumor regression in animal models. To investigate the antitumor effect of direct gene transfer of human IL-12 into tumors, human IL-12 p35 and p40 cDNAs were cloned into the Semliki Forest virus (SFV) vector pSFV1. In order to express the two subunits from the same vector, the p35 and the p40 cDNAs were cloned into pSFV1, each under the control of a subgenomic SFV promoter. Recombinant RNA produced by in vitro transcription of SFV-IL-12 construct, was packaged into SFV viral particles with the use of a non-packageable helper RNA. We show that human tumor cell lines infected in vitro in vivo with recombinant SFV-IL-12 viral particles secrete high levels of biologically active heterodimeric p35/p40 IL-12, as demonstrated using ELISA and biological assays.

Gene Expression↗
PubMed1996

CD40 engagement induces monocyte procoagulant activity through an interleukin-10 resistant pathway.

Interactions between membrane-bound molecules were previously shown to be involved in the induction of tissue factor-dependent monocyte procoagulant activity (PCA) by activated T cells. To investigate the potential role of the CD40/CD40 ligand (CD40L) pathway in this process, we first determined the effects of blocking anti-CD40 or anti-CD40L monoclonal antibodies (mAb) on the development of monocyte PCA during mixed lymphocyte reaction (MLR) between allogeneic peripheral blood mononuclear cells (PBMC). The strong inhibitory effect exerted by both mAb (mean percentages of inhibition: 88 and 91% for anti-CD40 and anti-CD40L mAb, respectively) indicates that CD40/CD40L interactions are required for the induction of PCA in MLR. These data led us to measure monocyte PCA after incubation of PBMC or purified monocytes with a stimulating anti-CD40 mAb (BL-C4) or with 3T6 fibroblasts transfected with the gene encoding CD40L. In both systems, we found that CD40 engagement strongly induced monocyte PCA which was related to tissue factor expression as shown by flow cytometric analysis. Finally, we observed that recombinant interleukin (IL)-10, which inhibits lipopolysaccharide-induced PCA, did not significantly influence CD40-dependent PCA. We conclude that CD40 engagement on monocytes induces tissue factor-dependent PCA through an IL-10-resistant pathway. These findings have implications for the control of coagulation events triggered by interactions between T cells and monocytes.

Antibodies, Monoclonal↗
PubMed1995

Interleukin-10 differentially regulates B7-1 (CD80) and B7-2 (CD86) expression on human peripheral blood dendritic cells.

Most of the immunosuppressive effects of interleukin-10 (IL-10) are related to functional inhibition of antigen-presenting cells (APC). Herein, we investigate the influence of recombinant (r)IL-10 on human dendritic cells (DC) purified from peripheral blood of healthy volunteers. First, we found that rIL-10 inhibited in a dose-dependent manner the proliferative responses as well as the production of IL-2 and interferon-gamma (IFN-gamma) in mixed lymphocyte reaction (MLR) between purified T cells and DC. This rIL-10 effect could be attributed to a direct effect on DC, as DC preincubated with rIL-10 were found to be deficient in the induction of alloreactive T cells even when anti-IL-10 neutralizing mAb was added at the time of MLR. Flow cytometric analysis indicated that rIL-10 did not modify the expression of ICAM-1 (CD54) and B7-1 (CD80), but decreased HLA-DR and B7-2 (CD86) expression at the DC surface. We conclude that the inhibitory effect of rIL-10 on primary alloreactive T cell responses involves down-regulation of class II MHC and B7-2 expression at the DC surface.

Antigens, CD↗
PubMed1995

The induction of human T cell unresponsiveness by soluble anti-CD3 mAb requires T cell activation.

In order to obtain an in vitro model of human T cell unresponsiveness induced by soluble anti-CD3 mAb in the presence of accessory cells, T cells purified from peripheral blood of healthy volunteers were cultured for 4 days with irradiated accessory cells and OKT3. After a 48 h resting period allowing TCR-CD3 complex re-expression, T cells were rechallenged with plastic-immobilized OKT3, and their proliferative response as well as their secretion of IL-2, IFN-gamma and IL-10 measured. Primary culture with OKT3 induced a state of unresponsiveness characterized by defective responses to OKT3 rechallenge but normal or enhanced responses to PMA and A23187 calcium ionophore, indicating a defect in the early steps of TCR-CD3-mediated signal transduction. Indeed, we found that unresponsive T cells displayed an impaired mobilization of intracellular calcium stores upon TCR-CD3 ligation. In order to determine whether the development of unresponsiveness depends on the initial T cell activation triggered by OKT3, we compared several versions of OKT3 differing in their ability to bind Fc receptors. We found that only the activating antibodies that bind Fc receptors on accessory cells induced T cell unresponsiveness. We conclude that human resting T cells can be rendered unresponsive by anti-CD3 mAb in soluble form provided that they trigger T cell activation.

Antigen-Presenting Cells↗
PubMed1994

B7/CD28-dependent IL-5 production by human resting T cells is inhibited by IL-10.

We analyzed the effects of rIL-10 on IL-5 production by human resting T cells isolated from peripheral blood. Resting T cells of healthy individuals required activation for 48 h with either anti-CD3 mAb cross-linked on B7/CD32-transfected mouse fibroblasts or PMA in conjunction with anti-CD28 mAb for optimal IL-5 secretion. In each condition, IL-5 secretion measured by ELISA was inhibited in a dose-dependent manner by rIL-10, whereas IFN-gamma production was not suppressed. The inhibitory effect of rIL-10 on IL-5 synthesis induced by PMA and anti-CD28 mAb was also observed at the mRNA level. In contrast with its action on T cells costimulated by B7/CD28 signaling, rIL-10 did not block IL-5 secretion in response to PMA and A23187 calcium ionophore. The inhibition of IL-5 production by rIL-10 was not due to IL-2 deprivation because it was not modified by the addition of exogenous rIL-2. Moreover, cyclosporin A, which inhibited IL-2 more efficiently than rIL-10 in response to anti-CD3 mAb and B7/CD32 transfected fibroblasts, did not reduce and even enhanced IL-5 production. Finally, we analyzed the influence of endogenously produced IL-10 on IL-5 secretion by T cells stimulated by PMA and anti-CD28 mAb. Addition of a neutralizing anti-IL-10 mAb increased IL-5 release in this system, indicating that endogenous IL-10 controls IL-5 production. We conclude that both rIL-10 and endogenous IL-10 inhibit IL-5 production by T cells costimulated by B7/CD28 signaling.

Animals↗
PubMed1994

Interleukin-10 inhibits B7 and intercellular adhesion molecule-1 expression on human monocytes.

There is evidence that interleukin -10(IL-10) interferes with the costimulatory properties of antigen-presenting cells and, thereby, inhibits their ability to induce T cell activation. To determine whether this effect might involve modulation of the expression of accessory molecules, we analyzed by flow cytometry the influence of human IL-10 on the basal expression of intercellular adhesion molecule 1 (ICAM-1) as well as on the interferon gamma (IFN-gamma)-induced up-regulation of ICAM-1 and B7 at the surface of human monocytes. IL-10 inhibited both the basal expression and the IFN-gamma-induced ICAM-1 up-regulation. IL-10 also reduced B7 up-regulation on IFN-gamma-stimulated monocytes. The inhibitory effect of IL-10 both on ICAM-1 and B7 expression was shown to be dose dependent. We conclude that the ability of IL-10 to decrease both ICAM-1 and B7 expression on monocytes might contribute to its immunosuppressive properties.

B7-1 Antigen↗