[Show abstract][Hide abstract] ABSTRACT: CCL18 is a human chemokine secreted by monocytes and dendritic cells. The receptor for CCL18 is not yet known and the functions of this chemokine on immune cells are not fully elucidated. In this study, we describe that CCL18 is present in skin biopsies of atopic dermatitis (AD) patients but not in normal or psoriatic skin. CCL18 was specifically expressed by APCs in the dermis and by Langerhans and inflammatory dendritic epidermal cells in the epidermis. In addition, the serum levels of CCL18 and the percentages of CCL18-producing monocyte/macrophages and dendritic cells were significantly increased in AD patients compared with healthy controls. Furthermore, we demonstrate that CCL18 binds to CLA(+) T cells in peripheral blood of AD patients and healthy individuals and induces migration of AD-derived memory T cells in vitro and in human skin-transplanted SCID mice. These findings highlight a unique role of CCL18 in AD and reveal a novel function of this chemokine mediating skin homing of a subpopulation of human memory T cells.
The Journal of Immunology 03/2005; 174(3):1723-8. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: p40, the common subunit of the proinflammatory cytokines IL-12 and IL-23, is produced by resident skin cells. Whereas the in vivo effects of IL-12 are well established, little is known about the role of IL-23 in cutaneous immune responses. In this study we show that p40 transgenic (TG) mice constitutively produce IL-23 (p19/p40), but not IL-12 (p35/p40), in basal keratinocytes by cosecretion of TG p40 with endogenous p19. Repeated injections of rIL-23 in littermate (LM) mice result in an inflammatory skin disease similar to that of p40 TG mice, confirming the proinflammatory activity of IL-23. Furthermore, IL-23 secretion by p40 TG keratinocytes induces elevated numbers of Langerhans cells (LC) with a marked up-regulation of costimulatory molecules, indicating advanced maturation of keratin 14 (K14)/p40 LC when compared with LM LC. At the functional level, freshly isolated K14/p40 LC greatly exceeded LC from LM animals in their capacity to stimulate allogeneic T cell proliferation. To assess whether IL-23 regulates cutaneous immune responses in vivo, we used an allogeneic skin transplantation model. Full thickness skin grafts from K14/p40 donors (H-2(q)) transplanted across a MHC class I and class II barrier onto BALB/c (H-2(d)) recipients were rejected in a significantly accelerated fashion (mean survival time: 8.8 days) when compared with skin grafts from non-TG LM (H-2(q)) (mean survival time: 10.7 days, p < 0.01). Based on these results we propose that IL-23-induced changes of LC may be an important mechanism in directing the outcome of cutaneous immune responses.
The Journal of Immunology 07/2003; 170(11):5438-44. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Myeloid lineage-derived dendritic cells (DCs) are considered the professional antigen-presenting cell type responsible for eliciting T-cell-mediated immune responses. Acute myelogenous leukemia (AML) is a disease in which tumor antigens are expressed by the malignant clone that also has the potential to differentiate into DC-like cells (leukemic DCs) with antigen-presenting capacity. This study investigated whether the constitutive expression of the cytokine interleukin-7 (IL-7) in primary AML cells during their differentiation toward leukemic DCs results in superior antigen-presenting cells. A bicistronic retroviral vector encoding the IL-7 cytokine and the surface immunoselectable low-affinity nerve growth factor receptor (LNGFr) gene was constructed and used for transduction experiments. A serum-free system was used to transduce and differentiate leukemic cells toward leukemic DCs. The study included 8 patients with AML. The transduction efficiency with the cytokine vector varied among patients, ranging from 5% to 30% as judged by LNGFr expression. The leukemic origin of the transduced cells was confirmed in a patient with a chromosomal translocation t(9:11) by fluorescence in situ hybridization analysis. Cytokine modified-cells consistently secreted IL-7 (mean, 415 pg +/- 190/10(6) cells/48 hours; n = 5). We demonstrate that IL-7-transduced cells are included in the differentiated leukemic DC subset, and, as shown in a particular case, that about half of the mature CD80(+) and CD83(+) populations coexpress the LNGFr transgene. In addition, IL-7-modified leukemic cells induce stronger allo-T-cell stimulation and higher amounts of IL-2 production in T cells compared with control groups. Finally, cytokine-transduced leukemic DCs can effectively prime and generate cytotoxic T lymphocytes against autologous leukemic blasts.
[Show abstract][Hide abstract] ABSTRACT: Expression of CD68 (macrosialin) in the absence of surface and lysosomal lineage marker molecules is a characteristic feature of T zone-associated plasmacytoid monocytes, which were recently shown to represent precursors of dendritic cells (DC). We demonstrate here a minor population of strongly CD68-positive (CD68bright) blood cells that lack all analyzed myeloid surface (CD14-, CD33-, CD13-, CD11b-, CD11c-) and lysosomal (myeloperoxidase, MPO- and lysozyme, LZ-) marker molecules (0.4 +/- 2% of the total mononuclear cells). These CD68bright, lineage marker-negative (lin-) cells can be induced to proliferate in the presence of IL-3. They do not acquire myeloid features even upon stimulation with granulocyte-macrophage CSF plus IL-1, IL-3, and IL-6. Instead, these cells develop typical DC characteristics upon culture. Furthermore, these CD68brightlin- DC precursors acquire mature DC characteristics (CD86+, CD83+, CD54bright) upon stimulation with CD40 ligand plus IL-3. A second subset of DC precursor-like blood cells was found to weakly express CD68 (0.3 +/- 0.2% of the total mononuclear cells) and to coexpress several myeloid lineage associated molecules (LZ+, CD11c+, CD33+, CD13+). Cells of this second subset resemble both previously described myeloid-related peripheral blood DC and germinal center DC. Analysis of peripheral blood leukocytes for CD68 thus revealed the existence of two cell subsets that phenotypically resemble lymphoid tissue-associated DC. The unique phenotype CD68brightlin- is highly reminiscent of T zone-associated plasmacytoid monocytes. CD68brightlin- blood leukocytes also functionally resemble plasmacytoid monocytes. The lack of all analyzed myeloid features by CD68brightlin- blood leukocytes suggests that these cells arise from a novel nonmyeloid human DC differentiation pathway.
The Journal of Immunology 08/1998; 161(2):740-8. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epidermal Langerhans cells (LC) play a critical role in host defense. Still we know rather little about the development and functional specialization of these bone marrow-derived dendritic cells (DC) located in the most peripheral ectodermal tissue of the mammalian organism. How LC develop from their primitive progenitors in bone marrow and to what extent LC are related in their development to other lineages of the hemopoietic system is still under debate. There are currently 3 major areas of debate: 1) which are the signals required for LC development and differentiation to occur, 2) what are the (molecular) characteristics of the intermediate stages of LC differentiation, and 3) how are LC related in their development and/or function to other cells of the hemopoietic system? A better understanding of LC development and answers to these questions can be expected from recently developed technologies which allow the in vitro generation of DC with the typical molecular, morphological and functional features of LC from purified CD34+ progenitor cells under defined serum-free culture conditions. TGF-beta 1 was found to be an absolute requirement for in vitro LC development under serum-free conditions upon stimulation with the classical DC growth and differentiation factors GM-CSF, TNF-alpha and SCF. The recently identified cytokine FLT3 ligand further dramatically enhanced in vitro LC development and even allowed efficient in vitro generation of LC colonies from serum-free single cell cultures of CD34+ hemopoietic progenitor cells.
[Show abstract][Hide abstract] ABSTRACT: The distribution of myeloid lineage-associated cytokine receptors and lysosomal proteins was analyzed in human CD34+ cord blood cell (CB) subsets at different stages of myeloid commitment by reverse-transcriptase polymerase chain reaction (RT-PCR). The highly specific granulomonocyte-associated lysosomal proteins myeloperoxidase (MPO) and lysozyme (LZ), as well as the transcription factor PU.1, were already detectable in the most immature CD34+Thy-1+ subset. Messenger RNA (mRNA) levels for the granulocyte-colony stimulating factor (G-CSF) receptor, granulocyte-macrophage (GM)-CSF receptor alpha subunit and tumor necrosis factor (TNF) receptors I (p55) and II (p75) were also detected in this subset in addition to c-kit and flt-3, receptors known to be expressed on progenitor cells. By contrast, the monocyte-macrophage colony stimulating factor (M-CSF) receptor was largely absent at this stage and in the CD34+Thy-1-CD45RA- subsets. The M-CSF receptor was first detectable in the myeloid-committed CD34+Thy-l-CD45RA+ subset. All other molecules studied were found to be expressed at this stage of differentiation. Different cocktails of the identified ligands were added to sorted CD34+Thy-1+ single cells. Low proliferative capacity was observed after 1 week in culture in the presence of stem cell factor (SCF) + Flt-3 ligand (FL) + G-CSF. Addition of GM-CSF to this basic cocktail consistently increased the clonogenic capacity of single CD34+Thy-1+ cells, and this effect was further enhanced (up to 72.3 +/- 4.3% on day 7) by the inclusion of TNF-alpha. In conclusion, the presence of myeloid-associated growth factor receptor transcripts in CD34+ CB subsets does not discriminate the various stages of differentiation, with the exception of the M-CSF receptor. In addition, we show that TNF-alpha is a potent costimulatory factor of the very immature CD34+Thy-1+ CB subset.
[Show abstract][Hide abstract] ABSTRACT: A retroviral-vector encoding the low affinity nerve growth factor receptor (LNGFR) was used to transduce dendritic cells (DCs) generated from CD34+ cord blood (CB) progenitor cells under serum-free conditions. Transduction efficiency was monitored by flow cytometry (FACS) using a specific monoclonal antibody. Prior to retroviral infections, CD34+ CB cells were stimulated for 60 h in a serum-free medium containing a DC differentiation inducing cytokine cocktail: stem cell factor (SCF), granulocyte/macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFalpha), and transforming growth factor beta 1 (TGF-beta1). Addition of flt3-ligand (FL) to the aforementioned growth factors significantly enhanced cell expansion (41.7+/-11.5 fold vs. 22.5+/-4.7 fold without FL) and generation of CD1a+ DCs (mean 45.7+/-9.8% vs. 28+/-6.5% without FL, n = 4,p = 0.01). Furthermore, FL significantly increased the proportion of CD1a+LNGFR+ cells (mean 10%+/-4.4% vs. 6%+/-2.4 without FL n = 4, p = 0.03). When serum-free viral supernatants were used to infect DCs progenitors under entirely serum-free conditions and with the most potent cytokine combination, approximately one-third of the CD1a+ DCs generated co-expressed the LNGFR gene. Moreover, the transduced gene was also identified in more mature CD1a+CD80+ and CD1a+CD86+ DCs after 12-14 days of culture. In addition, transduced CD1a+ DCs maintained their functional properties, stimulating allogeneic T cells with similar efficiency as nontransduced CD1a+ DCs. Thus, the serum-free system described allows efficient generation and transduction of CD1a+ DCs derived from CD34+ progenitor cells and may be very useful for future therapeutic applications of DCs.
Human Gene Therapy 10/1997; 8(14):1651-8. · 4.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using a recently described serum-free culture system of purified human CD34+ progenitor cells, we show here a critical cooperation of flt3 ligand (FL) with transforming growth factor-beta1 (TGF-beta1) in the induction of in vitro dendritic cell/Langerhans cell (DC/LC) development. The addition of FL to serum-free cultures of CD34+ cells supplemented with TGF-beta1, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha, and stem cell factor strongly increases both percentages (mean, 36% +/- 5% v 64% +/- 4%; P = .001) and total numbers (4.4- +/- 0.8-fold) of CD1a+ dendritic cells. These in vitro-generated CD1a+ cells molecularly closely resemble a particular type of DC known as an epidermal Langerhans cell. Generation of DC under serum-free conditions was found to strictly require supplementation of culture medium with TGF-beta1. Upon omission of TGF-beta1, percentages of CD1a+ DC decreased (to mean, 10% +/- 8%; P = .001) and, in turn, percentages of granulomonocytic cells (CD1a- cells that are lysozyme [LZ+]; myeloperoxidase [MPO+]; CD14+) increased approximately threefold (P < .05). Furthermore, in the absence of TGF-beta1, FL consistently promotes generation of LZ+, MPO+, and CD14+ cells, but not of CD1a+ cells. Serum-free single-cell cultures set up under identical TGF-beta1- and FL-supplemented culture conditions showed that high percentages of CD34+ cells (mean, 18% +/- 2%; n = 4) give rise to day-10 DC colony formation. The majority of cells in these DC-containing colonies expressed the Langerhans cell/Birbeck granule specific marker molecule Lag. Without TGF-beta1 supplementation, Lag+ colony formation is minimal and formation of monocyte/macrophage-containing colonies predominates. Total cloning efficiency in the absence and presence of TGF-beta1 is virtually identical (mean, 41% +/- 6% v 41% +/- 4%). Thus, FL has the potential to strongly stimulate DC/LC generation, but has a strict requirement for TGF-beta1 to show this costimulatory effect.
[Show abstract][Hide abstract] ABSTRACT: The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder described as a clinical triad of thrombocytopenia, eczema, and immunodeficiency. The gene responsible for WAS encodes a 502-amino acid proline-rich protein (WASp) that is likely to play a role in the cytoskeleton reorganization and/or in signal transduction of hematopoietic cells. However, the function and the regulation of the WAS gene (WASP) have not yet been clearly defined. We have studied WASP expression at the transcriptional level in freshly isolated mature peripheral blood cells and during hematopoietic development. For this purpose, we have isolated CD34+ hematopoietic precursor cells from cord blood. These cells were cultured in vitro with various growth factors to generate committed or mature cells belonging to different hematopoietic differentiation pathways, such as granulocytic (CD15+) cells, monocytic (CD14+) cells, dendritic (CD1a+) cells, erythroid lineage (glycophorin A+) cells, and megakaryocytic cells (CD41+). We have shown by reverse transcriptase polymerase chain reaction analysis that the WASP transcript is ubiquitously detectable throughout differentiation from early hematopoietic progenitors, including CD34+CD45RA- and CD34+CD45RA+ cells, to cells belonging to different hematopoietic lineages, including erythroid-committed and dendritic cells. In addition, Northern blot analysis showed that peripheral blood circulating lymphocytes (CD3+ and CD19+ cells) and monocytes express WASP mRNA. Several hematopoietic cell lines were tested and higher levels of expression were consistently detected in myelomonocytic cell types. By contrast, primary nonhematopoietic cells, including fibroblasts, endothelial cells, and keratinocytes, were consistently negative for WASP mRNA.
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DC) are the most potent APCs within the immune system. We show here that highly purified CD14(bright) peripheral blood monocytes supplemented with granulocyte-monocyte (GM)-CSF plus IL-4 develop with high efficacy (>95% of input cells) into DC. They neo-expressed CD1a, CD1b, CD1c, CD80, and CD5; they massively up-regulated CD40 (109-fold) and HLA-DQ and DP (125- and 87-fold); and significantly (>5-fold) up-regulated HLA-DR, CD4, CD11b, CD11c, CD43, CD45, CD45R0, CD54, CD58, and CD59. CD14, CD15s, CD64, and CDw65 molecules were down-regulated to background levels, and no major changes were observed for HLA class I, CD11a, CD32, CD33, CD48, CD50, CD86, CDw92, CD93, or CD97. Monocytes cultured in parallel with GM-CSF plus TNF-alpha were more heterogeneous in expression densities but otherwise similar in their surface molecule repertoire. They clearly differed, however, in their accessory cell capacity. Only GM-CSF plus IL-4-cultured cells were found to be potent stimulators in allogeneic and autologous MLR and they presented tetanus toxoid 100- to 1000-fold more efficiently than other cell populations tested. Furthermore, only cytokine-treated monocytes formed clusters with resting T cells. At variance from all these similarities between in vitro-generated monocyte-derived DC and in vivo-developing DC, the DC populations generated by us contained significant amounts of myeloperoxidase and also expressed lysozyme. At least in this respect they, thus, differ from "classical" DC types.
The Journal of Immunology 12/1996; 157(9):3850-9. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several studies have demonstrated that dendritic cells (DC) can be generated in vitro from CD34+ hemopoietic progenitor cells. The growth requirements for these cells are poorly characterized, however. In particular, undefined serum/plasma components seem to significantly contribute to in vitro DC development. We report here that the cytokine combination granulocyte-macrophage CSF (GM-CSF) plus TNF-alpha and stem cell factor (SCF) commonly used for the in vitro generation of DC in serum/plasma-supplemented medium is, in the absence of serum supplementation, very inefficient in inducing DC development. We further demonstrate that supplementation with TGF-beta 1 is required for substantial DC development to occur in the absence of serum. Culture of CD34+ cells under serum-free conditions with TGF-beta 1 plus GM-CSF, TNF-alpha, and SCF strongly induces DC differentiation. This culture condition is even more efficient than culturing CD34+ cells with GM-CSF plus TNF-alpha and SCF in the presence of cord blood plasma. The proportions and total yields of cells with typical DC morphology and CD1a molecule expression are higher. The allostimulatory capacity of DC from TGF-beta 1-supplemented, cultures exceeds allostimulation by cells grown in plasma-containing medium. Substantial numbers (21 +/- 7%) of cells grown in TGF-beta 1-supplemented, but not plasma-supplemented, cultures express the Birbeck granule marker molecule Lag and display numerous Birbeck granules. Cells with distinct monocytic features are less frequently observed in TGF-beta 1-supplemented serum-free cultures. The addition of neutralizing anti-TGF-beta 1 Ab abrogates the observed TGF-beta 1 effects.
The Journal of Immunology 09/1996; 157(4):1499-507. · 5.52 Impact Factor