Human cytomegalovirus-derived protein UL18 alters the phenotype and function of monocyte-derived dendritic cells.

Human cytomegalovirus (HCMV) encodes the MHC class I-like molecule UL18, which binds with high affinity to the leukocyte Ig-like receptor-1 (LIR-1), an inhibitory receptor commonly expressed on myeloid cells and subsets of NK and T cells. The exact role of UL18 is not known, in particular in relation to its proposed role in HCMV immune escape. Given the ubiquitous expression of LIR-1 on dendritic cells (DCs), we hypothesized that UL18 may affect DC function. To study the effects of UL18 on DC, we made use of UL18 fusion proteins. We demonstrate that UL18 fusion proteins inhibit the chemotaxis of DCs. Furthermore, UL18 interfered with CD40 ligand-induced maturation of DCs, resulting in reduced allogeneic T cell proliferation. Finally, we demonstrate that UL18 proteins up-regulate the expression of the maturation marker CD83 on immature monocyte-derived DCs and induce cytokine production. The capacity of UL18 to affect the function and the phenotype of DCs suggests a novel role for this HCMV-derived protein.

Dexamethasone-induced apoptosis in acute lymphoblastic leukemia involves differential regulation of Bcl-2 family members.

BACKGROUND AND OBJECTIVES: The mechanism of glucocorticoid -induced apoptosis is not fully understood and early predictive assays based on apoptotic markers for clinical outcome in acute lymphoblastic leukemia (ALL) are scarce. The aim of this study was to characterize the involvement of Bcl-2 family members and caspase activation in dexamethasone(Dex)-induced apoptosis in ALL. DESIGN AND METHODS: Primary childhood ALL samples, the pre-B ALL cell line RS(4;11), and the T-ALL cell line CCRF-CEM were used. The involvement of Bcl-2 family members was evaluated by flow cytometry, immunocytochemistry, and western and northern blotting. Apoptosis was analyzed by annexin V and TMRE staining. Caspase activity was evaluated by a fluorometric assay. RESULTS: Dex induced significant down-regulation of the anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL, differential activation of the pro-apoptotic Bak and Bax, loss of Delta psi m and cytochrome c release. Dex-induced apoptosis also involved early activation of caspases 2 and -3. Inhibition of caspase activity did not, however, protect against Dex-induced Bak/Bax activation, loss of Delta psi m or cell death. In 12 primary ALL samples Dex-induced apoptosis was associated with activation of Bax (p=0.045) and down-regulation of Bcl-2 (p=0.016) and/or Bcl-xL (p=0.004). Furthermore, ex vivo Dex-sensitivity was associated with an early treatment response to polychemotherapy (p=0.026). INTERPRETATION AND CONCLUSIONS: The differential regulation of pro- and anti-apoptotic Bcl-2 family members appears to be a key event in the execution of Dex-induced apoptosis in ALL cell lines, and also indicates a role for these proteins in primary ALL cells.

Do CD8 effector cells need IL-7R expression to become resting memory cells?

The role for IL-7R expression in the differentiation of effector T cells into resting memory remains controversial. Here, using a conditional IL-7R transgenic model, we were able to test directly whether CD8 effector T cells require IL-7R expression for their differentiation into resting memory cells. In the absence of IL-7R expression, effector cells transferred into "full" hosts underwent a protracted and unremitting contraction compared with IL-7R-expressing control cells and were unable to develop into long-term resting memory cells. Surprisingly, when the same effector cells were transferred into empty T-cell-deficient hosts, they could generate long-lived fully functional resting memory cells independently of IL-7R expression. Formation of these latter cells was found to be dependent on IL-15, because the same IL-7R-deficient effector cells were rapidly lost from IL-15-deficient hosts, having a half-life of less than 40 hours. Therefore, our data suggest that, under physiological conditions, both IL-7 and IL-15 synergize to promote the formation of memory cells directly by limiting the contraction of effectors that occurs following an immune response and that reexpression of IL-7R is a key checkpoint in the regulation of this process.

Functional interaction of CARD15/NOD2 and Crohn's disease-associated TNFalpha polymorphisms.

BACKGROUND AND AIMS: Mutations/polymorphisms in the CARD15/NOD2 gene and in the promoter region of the TNFalpha gene are associated with susceptibility to and modulate the phenotype of Crohn's disease (CD). The molecular mechanisms for this genotype-phenotype correlation are yet to be elucidated. CARD15 is an intracellular receptor for bacterial muramyl dipeptide (MDP), and can elicit an inflammatory response via activation of the NF-kappaB pathway. MDP is also known to induce the expression of pro-inflammatory cytokines including TNFalpha, through a still poorly characterized signaling pathway. We sought to determine whether CARD15-mediated NF-kappaB activation can contribute to MDP-induced TNFalpha production and, consequently, if polymorphisms in both genes affect the control of such induction. METHODS/RESULTS: Transfection and electrophoretic mobility shift assays (EMSA) experiments in HEK293 cells demonstrated that MDP exposure stimulates TNFalpha gene transcription, as a result of CARD15-induced NF-kappaB activation and binding to TNFalpha promoter. When the CD-associated CARD15 1007fs variant was analyzed, induction of TNFalpha promoter activity was found to be defective. Different combinations of CARD15 and TNFalpha promoter polymorphisms gave rise to distinct TNFalpha transcription levels. CONCLUSIONS: CARD15 and TNFalpha promoter polymorphisms interact to exert a functional effect on MDP-induced TNFalpha production. This gene-gene interaction may contribute to interindividual variation in susceptibility to, and manifestation of, Crohn's disease.

Malassezia sympodialis stimulation differently affects gene expression in dendritic cells from atopic dermatitis patients and healthy individuals.

It is known that 28-84% of patients with atopic dermatitis exhibit IgE and/or T-cell reactivity to the opportunistic yeast Malassezia sympodialis, which can be taken up by immature monocyte-derived dendritic cells (MDDCs), resulting in MDDC maturation. The aim of this study was to investigate whether MDDCs from patients with atopic dermatitis respond differently to M. sympodialis compared to MDDCs from healthy individuals. Immature MDDCs were stimulated with M. sympodialis and the gene expression profiles were analysed with cDNA arrays containing 406 genes. Our results show that M. sympodialis differently affected MDDCs from patients with atopic dermatitis, and more so in severely ill patients, compared with healthy individuals. Six genes were more than fivefold up-regulated in MDDCs from more than one patient with atopic dermatitis, coding for CD54, CD83, IL-8, monocyte-derived chemokine (MDC), BTG1 and IL-1R antagonist. In healthy individuals this was true only for BTG1. Up-regulations of IL-8 and MDC were confirmed at the protein level. Our findings might reflect an increased trafficking and stimulatory capacity in MDDCs from the patients, which is likely to result in a stronger inflammatory response to M. sympodialis.

A novel adjuvant-allergen complex, CBP-rFel d 1, induces up-regulation of CD86 expression and enhances cytokine release by human dendritic cells in vitro.

Allergen-specific immunotherapy is commonly performed with allergen extracts adsorbed to aluminium hydroxide (alum). The undesirable effects associated with the use of alum, including granuloma formation at the site of injection and stimulation of T helper 2 (Th2) cytokine production, has generated interest in alternative allergen carriers, one being carbohydrate-based particles (CBPs). Here, we have investigated the in vitro effects of the recombinant major cat allergen Fel d 1 (rFel d 1) coupled to CBPs (CBP-rFel d 1) on human monocyte-derived dendritic cells (MDDCs) obtained from healthy blood donors. A majority of the CD1a(+) MDDCs internalized fluorescein isothiocyanate-labelled CBP-rFel d 1, as demonstrated by flow cytometry and confocal laser-scanning microscopy. Furthermore, an up-regulation of the expression of the costimulatory molecule, CD86, on the MDDCs was induced by CBP-rFel d 1, but not by rFel d 1 or CBPs alone. Finally, three- and fourfold increases in the release of interleukin-8 and tumour necrosis factor-alpha, respectively, were observed when MDDCs were cultured in the presence of CBP-rFel d 1. Altogether, our results indicate that the use of CBPs as an allergen carrier and adjuvant is a promising candidate for the improvement of allergen-specific immunotherapy.

Microvilli structures on B lymphocytes: inducible functional domains?

Interactive contact between B lymphocytes and T cells is necessary for their expansion during an immune response. It has been shown that B lymphocytes receive signals from T cells, such as IL-4 and cross-linking of CD40, which are crucial for their differentiation. We previously found that these factors induce formation of microvilli on B cells and that this was correlated with increased homotypic adhesion of B lymphocytes. In this study we have investigated if IL-4 induce segregation of proteins to microvilli and lipid rafts. Using immuno-electron microscopy we analyzed cell-surface distribution of molecules involved in B-T cell co-activation. Recruitment to detergent-resistant membrane fractions was analyzed using sucrose gradient centrifugation. We found that microvilli were enriched in ICAM-1 and MHC class II molecules. In contrast, LFA-1 and CD40 were more abundant on the smooth cell surfaces, while B7-2 (CD86) was randomly distributed. We also discovered that depletion of cholesterol, using beta-methyl-cyclodextrin, lowered the number of microvilli, indicating that intact lipid rafts are required for their expression. Moreover, activation of B lymphocytes by lipopolysaccharide (LPS) induced increased expression of GM(1), a marker for lipid rafts. However, although both surface and total levels of GM(1) were similar in B lymphocytes stimulated with either LPS or LPS plus IL-4, GM(1) was mainly expressed on microvilli in LPS plus IL-4-stimulated cells. Taken together, our results indicate that microvilli represent distinct inducible membrane domains that can regulate direct cell-cell interactions via grouping and three-dimensional presentation of cell-surface receptors.

Dendritic cells and fungi.

Fungi comprise a group of microorganisms that in the past 20 years has become increasingly important as a cause of human disease. Few fungi are professional but instead opportunistic pathogens, and some fungi can even act as allergens. Dendritic antigen-presenting cells function as a link between innate and adaptive immunity and are therefore important in recognition of pathogens. Effective defense requires the host to discriminate between different pathogens to induce an appropriate response. Signaling from different groups of microbes can be mediated via the Toll-like receptors (TLRs), leading to activation of conserved host defense signaling pathways that control the expression of a variety of immune response genes. Different dendritic cells (DCs) express different patterns of recognition molecules, which indicate that they are more or less efficient when responding to certain pathogens. DCs have an important role in the induction of cell-mediated immune responses to fungi, and the studies reviewed here show that fungi, or possibly fungi-derived factors, provide a powerful activation stimulus to DCs, resulting in DC maturation with upregulation of co-stimulatory molecules and production of cytokine patterns leading to different T cell responses. The possibility of using ex vivo-generated DCs as therapeutic tools for restoring anti-fungal immunity is a challenge for the future.

Natural killer and dendritic cell contact in lesional atopic dermatitis skin--Malassezia-influenced cell interaction.

The regulation of dendritic cells is far from fully understood. Interestingly, several recent reports have suggested a role for natural killer cells in affecting dendritic cell maturation and function upon direct contact between the cells. It is not known if this interaction takes place also in vivo, or if a potential interaction of natural killer cells and dendritic cells would be affected by allergen exposure of the dendritic cells. The yeast Malassezia can act as an allergen in atopic eczema/dermatitis syndrome, and induce maturation of dendritic cells. Our aims were to study the distribution of natural killer cells in the skin from atopic eczema/dermatitis syndrome patients with the emphasis on possible natural killer cell-dendritic cell interaction, and to assess whether the interaction of Malassezia with dendritic cells would affect subsequent interaction between dendritic cells and natural killer cells. A few scattered natural killer (CD56+/CD3-) cells were found in the dermis of healthy individuals and in nonlesional skin from atopic eczema/dermatitis syndrome patients. In lesional skin and in biopsies from Malassezia atopy-patch-test-positive skin, however, natural killer cells were differentially distributed and for the first time we could show close contact between natural killer cells and CD1a+ dendritic cells. Dendritic cells preincubated with Malassezia became less susceptible to natural-killer-cell-induced cell death, suggesting a direct effect imposed by Malassezia upon interaction of dendritic cells with natural killer cells. These findings indicate that natural killer cells and dendritic cells can interact in the skin and that Malassezia affects the interaction between natural killer cells and dendritic cells. Our data suggest that natural killer cells may play a role in regulating dendritic cells in atopic eczema/dermatitis syndrome.

Atopic eczema/dermatitis syndrome and Malassezia.

Atopic dermatitis is a chronic multifactorial inflammatory skin disease, which has had a marked increase in prevalence during the last decades. Recently, a new nomenclature was recommended where the term 'atopic eczema/dermatitis syndrome' (AEDS) should be used to reflect the heterogeneity in this group of patients and where those patients without measurable IgE reactivity should be classified as either 'nonallergic AEDS' or 'non-IgE-associated allergic AEDS'. For nearly 20 years it has been discussed whether the opportunistic yeast Malassezia, previously designated Pityrosporum, is a contributing factor to AEDS. Today there are several reports that demonstrate specific serum IgE or positive skin prick test and/or atopy patch test reactions to Malassezia in patients with AEDS. Several IgE-binding components have been identified in extracts of Malassezia ranging in molecular mass between 10 and 100 kD. The genes for nine Malassezia allergens with molecular weights ranging from 14 to 36 kD have hitherto been identified and cloned. Six of them are now produced by recombinant techniques and used in diagnostic tests. At present the genus Malassezia is subdivided into seven different species, which all have been isolated from human skin. The respective contribution of different Malassezia spp. to AEDS and in what proportion they share allergens remains to be clarified. We summarize here data that Malassezia can play a role in eliciting and maintaining eczema in patients with AEDS.