Christine Heufler

Production of IL-12 by Human Monocyte-Derived Dendritic Cells Is Optimal When the Stimulus Is Given at the Onset of Maturation, and Is Further Enhanced by IL-4

Dendritic cells produce IL-12 both in response to microbial stimuli and to T cells, and can thus skew T cell reactivity toward a Th1 pattern. We investigated the capacity of dendritic cells to elaborate IL-12 with special regard to their state of maturation, different maturation stimuli, and its regulation by Th1/Th2-influencing cytokines. Monocyte-derived dendritic cells were generated with GM-CSF and IL-4 for 7 days, followed by another 3 days ± monocyte-conditioned media, yielding mature (CD83+/dendritic cell-lysosome-associated membrane glycoprotein+) and immature (CD83−/dendritic cell-lysosome-associated membrane glycoprotein−) dendritic cells. These dendritic cells were stimulated for another 48 h, and IL-12 p70 was measured by ELISA. We found the following: 1) Immature dendritic cells stimulated with CD154/CD40 ligand or bacteria (both of which concurrently also induced maturation) secreted always more IL-12 than already mature dendritic cells. Mature CD154-stimulated dendritic cells still made significant levels (up to 4 ng/ml). 2) Terminally mature skin-derived dendritic cells did not make any IL-12 in response to these stimuli. 3) Appropriate maturation stimuli are required for IL-12 production: CD40 ligation and bacteria are sufficient; monocyte-conditioned media are not. 4) Unexpectedly, IL-4 markedly increased the amount of IL-12 produced by both immature and mature dendritic cells, when present during stimulation. 5) IL-10 inhibited the production of IL-12. Our results, employing a cell culture system that is now being widely used in immunotherapy, extend prior data that IL-12 is produced most abundantly by dendritic cells that are beginning to respond to maturation stimuli. Surprisingly, IL-12 is only elicited by select maturation stimuli, but can be markedly enhanced by the addition of the Th2 cytokine, IL-4.

The immunomodulator FTY720 interferes with effector functions of human monocyte-derived dendritic cells

The potent immunomodulator FTY720 elicits immunosuppression via acting on sphingosine 1‐phosphate receptors (S1PR), thereby leading to an entrapment of lymphocytes in the secondary lymphoid tissue. To elucidate the potential in vitro effects of this drug on human monocyte‐derived DC, we used low nanomolar therapeutic concentrations of FTY720 and phosphorylated FTY720 (FTY720‐P) and investigated their influence on DC surface marker expression, protein levels of S1PR and DC effector functions: antigen uptake, chemotaxis, cytokine production, allostimulatory and Th‐priming capacity. We report that both FTY720 and FTY720‐P reduce chemotaxis of immature and mature DC. Mature DC generated in the presence of FTY720 or FTY720‐P showed an impaired immunostimmulatory capacity and reduced IL‐12 but increased IL‐10 production. T cells cultured in the presence of FTY720‐ or FTY720‐P‐treated DC showed an altered cytokine production profile indicating a shift from Th1 toward Th2 differentiation. In treated immature and mature DC, expression levels for two S1PR proteins, S1P1 and S1P4, were reduced. We conclude that in vitro treatment with FTY720 affects DC features that are essential for serving their role as antigen‐presenting cells. This might represent a new aspect of the overall immunosuppressive action of FTY720 and makes DC potential targets of further sphingolipid‐derived drugs.

A Novel Role for IL-3: Human Monocytes Cultured in the Presence of IL-3 and IL-4 Differentiate into Dendritic Cells That Produce Less IL-12 and Shift Th Cell Responses Toward a Th2 Cytokine Pattern

Dendritic cells (DC) derived from plasmacytoid precursors depend on IL-3 for survival and proliferation in culture, and they induce preferentially Th2 responses. Monocytes express not only GM-CSF receptors, but also IL-3Rs. Therefore, we examined whether IL-3 had an effect on the functional plasticity of human monocyte-derived DC generated in a cell culture system that is widely used in immunotherapy. DC were generated with IL-3 (instead of GM-CSF) and IL-4. Yields, maturation, phenotype (surface markers and Toll-like receptors), morphology, and immunostimulatory capacity were similar. Only CD1a was differentially expressed, being absent on IL-3-treated DC. In response to CD40 ligation DC generated in the presence of IL-3 secreted significantly less IL-12 p70 and more IL-10 compared with DC grown with GM-CSF. Coculture of naive allogeneic CD4+ T cells with DC generated in the presence of IL-3 induced T cells to produce significantly more IL-5 and IL-4 and less IFN-γ compared with stimulation with DC generated with GM-CSF. These data extend the evidence that different cytokine environments during differentiation of monocyte-derived DC can modify their Th cell-inducing properties. A hitherto unrecognized effect of IL-3 on DC was defined, namely suppression of IL-12 secretion and a resulting shift from Th1 toward Th2.

Cloning, recombinant expression and biochemical characterization of the murine CD83 molecule which is specifically upregulated during dendritic cell maturation

Human CD83 (hCD83) is a glycoprotein expressed predominantly on the surface of dendritic cells (DC) and represents the best marker for mature DC. Here, we report the cloning of the cDNA encoding mouse CD83 (mCD83) from a murine bone marrow‐derived DC (BM‐DC) cDNA library. DNA sequence analysis revealed a 196 amino acid protein including a signal peptide of 21 amino acids which shares 63% amino acid identity with hCD83. Using Northern blot analyses, mCD83 mRNA was found to be strongly expressed in mouse BM‐DC and its expression was upregulated following stimulation with LPS or TNF‐α. Transfection experiments using COS‐7 cells revealed that mCD83 is glycosylated. Furthermore, the extracellular CD83 domain was recombinantly expressed in Escherichia coli and one‐dimensional NMR data strongly support that the protein is structurally folded.

Attenuation of cell adhesion in lymphocytes is regulated by CYTIP, a protein which mediates signal complex sequestration

An important theme in molecular cell biology is the regulation of protein recruitment to the plasma membrane. Fundamental biological processes such as proliferation, differentiation or leukocyte functions are initiated and controlled through the reversible binding of signaling proteins to phosphorylated membrane components. This is mediated by specialized interaction modules, such as SH2 and PH domains. Cytohesin‐1 is an intracellular guanine nucleotide exchange factor, which regulates leukocyte adhesion. The activity of cytohesin‐1 is controlled by phospho inositide‐dependent membrane recruitment. An interacting protein was identified, the expression of which is upregulated by cytokines in hematopoietic cells. This molecule, CYTIP, is also recruited to the cell cortex by integrin signaling via its PDZ domain. However, stimulation of Jurkat cells with phorbol ester results in re‐localization of CYTIP to the cytoplasm, and membrane detachment of cytohesin‐1 strictly requires co‐expression of CYTIP. Con sequently, stimulated adhesion of Jurkat cells to intracellular adhesion molecule‐1 is repressed by CYTIP. These findings outline a novel mechanism of signal chain abrogation through sequestration of a limiting component by specific protein–protein interactions.

Cross reactivity of three T cell attracting murine chemokines stimulating the CXC chemokine receptor CXCR3 and their induction in cultured cells and during allograft rejection

Recent work identified the murine gene homologous to the human T cell attracting chemokine CXC receptor ligand 11 (CXCL11, also termed I‐TAC, SCYB11, ß‐R1, H174, IP‐9). Here, the biological activity and expression patterns of murine CXCL11 relative to CXCL9 (MIG) and CXCL10 (IP‐10/crg‐2), the other two CXCR3 ligands, were assessed. Calcium mobilization and chemotaxis experiments demonstrated that murine CXCL11 stimulated murine CXCR3 at much lower doses than murine CXCL9 or murine CXCL10. Murine CXCL11 also evoked calcium mobilization in CHO cells transfected with human CXCR3 and was chemotactic for CXCR3‐expressing human T lymphocytes as well as for 300–19 pre‐B cells transfected with human or murine CXCR3. Moreover, murine CXCL11 blocked the chemotactic effect of human CXCL11 on human CXCR3 transfectants. Depending on cell type (macrophage‐like cells RAW264.7, J774A.1, fetal F20 and adult dermal fibroblasts, immature and mature bone marrow‐derived dendritic cells) andstimulus (interferons, LPS, IL‐1β and TNF‐α), an up to 10,000‐fold increase of CXCL9, CXCL10 and CXCL11 mRNA levels, quantified by real‐time PCR, was observed. In vivo, the three chemokines are constitutively expressed in various tissues from healthy BALB/c mice and were strongly up‐regulated during rejection of allogeneic heart transplants. Chemokine mRNA levels exceeded those of CXCR3 and IFN‐γ which were induced with similar kinetics by several orders of magnitude.

Sphingosine-1-phosphate receptor type-1 agonism impairs blood dendritic cell chemotaxis and skin dendritic cell migration to lymph nodes under inflammatory conditions

SEW2871 is a potent sphingosine-1-phosphate receptor type-1 (S1P(1))-selective agonist that induces peripheral lymphopenia through sequestration of lymphocytes into secondary lymphoid organs, similar to the non-selective sphingosine-1-phosphate (S1P) receptor agonist FTY720. FTY720 has been reported to interfere with human dendritic cell (DC) effector functions and both FTY720 and SEW2871 have been shown to modulate murine DC trafficking in vivo. Little is known about the possible effects of SEW2871 on human and murine DC functions. Here, we demonstrate that in contrast to FTY720, SEW2871 does not induce down-regulation of S1P(1) in human DCs and thus does not exert a functional antagonism at S1P(1). Notably, the compound was found to impair chemotaxis of immature and mature human DCs in vitro, possibly by interfering with the activation of p44/p42 and p38 mitogen-activated protein kinase signaling pathways. Comparative FACS analyses show that SEW2871 mediates CD18 down-regulation on mature human DCs. The influence on DC migration could be confirmed with in vivo assays using BALB/c mice in which SEW2871 impairs the migration of CD11c+ DC and CD207+ Langerhans cells (LC) to the draining lymph nodes (LNs) under inflammatory conditions. These results suggest that the S1P-S1P(1) axis might not only control lymphocyte trafficking but also play a pivotal role in DC migration from the skin to LN.

Generation of large numbers of human dendritic cells from whole blood passaged through leukocyte removal filters: an alternative to standard buffy coats.

Many blood banks now use whole blood inline filtration to produce leukocyte-depleted blood products. As a result, a common source of large numbers of human dendritic cells (DC) for research purposes, namely standard buffy coats, has been lost. Therefore, we have adapted our conventional method for growing DC from CD14(+) precursors in order to make use of these filter units. A dextran solution containing human serum albumin was used to flush back the filters. After pelleting, mononuclear cells were obtained by standard density gradient centrifugation (Lymphoprep). To eliminate T cells, we used rosetting with sheep red blood cells. In addition to the classical PBMC, the cell population obtained after Lymphoprep centrifugation was found to contain high numbers of CD14(+) granulocytes which could be depleted by separation on an additional Percoll gradient. At this stage, FACS analysis revealed a cell population that resembled the CD14(+) monocyte-enriched population, obtained from traditional buffy coat preparations after Lymphoprep centrifugation and T cell elimination. Culture of the cells and the induction of maturation was identical to the previously described procedures, except that the culture time was reduced from 7 to 5 days and the maturation time from 3 to 2 days. Analyses of the major molecules indicative of DC maturation (CD83, CD86, CD208/DC-LAMP) and functional analyses of the T cell-stimulatory capacity of the DC population (using the MLR assay with normal peripheral T cells and naive T cells) revealed no major differences from buffy coat-derived DC preparations.

Dendritic Cell Production of Cytokines and Responses to Cytokines

Dendritic cells (DC) are a family of bone marrow-derived MHC class II expressing cells which occur in small numbers in most lymphoid and nonlymphoid tissues. They represent a distinct lineage of leukocytes which can be found in two distinct maturational stages: immature dendritic cells are exemplified by the Langerhans cells in the epidermis, and are considered to be precursors to the mature dendritic cells in the lymphoid organs. These maturational stages can be distinguished by phenotypic and functional characteristics. Immature dendritic cells are weak stimulators of resting T lymphocytes but are excellent in processing soluble protein antigens for presentation to T cell clones. Mature dendritic cells show exactly reciprocal features. In this review the relatively few available data on cytokine production by DC and responses of DC to cytokines are collected. Our goal is to consider the role of cytokines in DC function including the transition from immature to mature stages.

Low tetrahydrobiopterin biosynthetic capacity of human monocytes is caused by exon skipping in 6-pyruvoyl tetrahydropterin synthase

Biosynthesis of (6 R )-5,6,7,8-tetrahydro-L-biopterin (H(4)-biopterin), an essential cofactor for aromatic amino acid hydroxylases and NO synthases, is effectively induced by cytokines in most of the cell types. However, human monocytes/macrophages form only a little H(4)-biopterin, but release neopterin/7,8-dihydroneopterin instead. Whereas 6-pyruvoyl tetrahydropterin synthase (PTPS) activity, the second enzyme of H(4)-biopterin biosynthesis, is hardly detectable in these cells, PTPS mRNA levels were comparable with those of cell types containing intact PTPS activity. By screening a THP-1 cDNA library, we identified clones encoding the entire open reading frame (642 bp) as well as clones lacking the 23 bp exon 3, which results in a premature stop codon. Quantification of the two mRNA species in different cell types (blood-derived cells, fibroblasts and endothelial cells) and cell lines showed that the amount of exon-3-containing mRNA is correlated closely to PTPS activity. The ratio of exon-3-containing to exon-3-lacking PTPS mRNA is not affected by differential mRNA stability or nonsense-mediated mRNA decay. THP-1 cells transduced with wild-type PTPS cDNA produced H(4)-biopterin levels and expressed PTPS activities and protein amounts comparable with those of fibroblasts. We therefore conclude that exon 3 skipping in transcription rather than post-transcriptional mechanisms is a major cause of the low PTPS protein expression observed in human macrophages and related cell types.