Transforming Growth Factor-β1 Regulates the Expression of the High-Affinity Receptor for IgE on CD34+ Stem Cell-Derived CD1a+ Dendritic Cells In Vitro
2004; Elsevier BV; Volume: 123; Issue: 4 Linguagem: Inglês
10.1111/j.0022-202x.2004.23428.x
ISSN1523-1747
AutoresJean‐Pierre Allam, Elisabeth Klein, Thomas Bieber, Natalija Novak,
Tópico(s)Immune Cell Function and Interaction
ResumoIt has been reported that monocytes, Langerhans cells (LC) and other dendritic cells (DC) express the high-affinity receptor for IgE (FcεRI) in patients with atopic diseases. These cells may be instrumental in the control of the immune response and the allergic inflammation. In this context, transforming growth factor beta 1 (TGF-β1) has been highlighted as a key cytokine involved in the mechanisms aimed to orchestrate tolerance and has been suggested as a candidate gene in atopic diseases. In this report, we investigate the putative role of TGF-β1 in the regulation of FcεRI on cord blood CD34+ stem cell-derived CD1a+ DC (CD34-derived CD1a+ DC). Kinetic experiments show that FcεRI spontaneously appears on the surface of CD1a+ DC, but decreases when exogenous TGF-β1 is added at high doses (10 ng per mL) or when endogenous TGF-β1 is neutralized in the culture conditions. In contrast, low-dose TGF-β1 (0.5 ng per mL) stabilizes surface FcεRI expression on DC. Increasing TGF-β1 concentrations leads to the generation of LC-like DC showing an augmentation in stimulatory capacity towards allogeneic T cells. In view of these data, a picture emerges that FcεRI+ on DC is finely modified by the TGF-β1 concentration in the microenvironment and could be of primary relevance in the context of atopic diseases. It has been reported that monocytes, Langerhans cells (LC) and other dendritic cells (DC) express the high-affinity receptor for IgE (FcεRI) in patients with atopic diseases. These cells may be instrumental in the control of the immune response and the allergic inflammation. In this context, transforming growth factor beta 1 (TGF-β1) has been highlighted as a key cytokine involved in the mechanisms aimed to orchestrate tolerance and has been suggested as a candidate gene in atopic diseases. In this report, we investigate the putative role of TGF-β1 in the regulation of FcεRI on cord blood CD34+ stem cell-derived CD1a+ DC (CD34-derived CD1a+ DC). Kinetic experiments show that FcεRI spontaneously appears on the surface of CD1a+ DC, but decreases when exogenous TGF-β1 is added at high doses (10 ng per mL) or when endogenous TGF-β1 is neutralized in the culture conditions. In contrast, low-dose TGF-β1 (0.5 ng per mL) stabilizes surface FcεRI expression on DC. Increasing TGF-β1 concentrations leads to the generation of LC-like DC showing an augmentation in stimulatory capacity towards allogeneic T cells. In view of these data, a picture emerges that FcεRI+ on DC is finely modified by the TGF-β1 concentration in the microenvironment and could be of primary relevance in the context of atopic diseases. 7-aminoactinomycin-D antigen-presenting cells dendritic cells fetal calf serum high-affinity IgE receptor alpha-chain of the high-affinity IgE receptor gamma-chain of the high-affinity IgE receptor Langerhans cells major histocompatibiliy complex I major histocompatibility complex II phycoerythrin transforming growth factor beta 1 The identification of the high-affinity receptor for IgE (FcεRI) on the surface of professional antigen-presenting cells (APC) from the myeloid lineage such as monocytes (Mo) and dendritic cells (DC) in the blood or tissue of atopic individuals lead to a new view of cellular distribution and functionality of this structure (Bieber, 1997Bieber T. Fc epsilon RI-expressing antigen-presenting cells: New players in the atopic game.Immunol Today. 1997; 18: 311-313Abstract Full Text PDF PubMed Scopus (90) Google Scholar). Interestingly, in contrast to basophils and mast cells, FcεRI surface expression on Mo and DC is highly variable. Therefore, factors regulating the FcεRI surface expression are of primary interest since they are crucial for our understanding of the physiological and pathophysiological role of this structure. In the last decade, much progress has been made in the development of in vitro models for the generation of large numbers of DC. The first strategy is based on the generation of so-called Mo-derived DC. Alternatively, DC can be derived from CD34+ stem cells obtained from human adult or cord blood. Detailed analysis revealed that under appropiate conditions, CD34+-derived cells have the capacity to acquire distinct properties of DC subtypes (Caux et al., 1992Caux C. Dezutter-Dambuyant C. Schmitt D. Banchereau J. GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells.Nature. 1992; 360: 258-261Crossref PubMed Scopus (1441) Google Scholar;Banchereau and Steinman, 1998Banchereau J. Steinman R.M. Dendritic cells and the control of immunity.Nature. 1998; 392: 245-252Crossref PubMed Scopus (12257) Google Scholar). In previous studies, a varying spontaneous surface expression of FcεRI on in vitro generated CD1a+ DC has been reported (Geiger et al., 2000Geiger E. Magerstaedt R. Wessendorf J.H. Kraft S. Hanau D. Bieber T. IL-4 induces the intracellular expression of the alpha chain of the high-affinity receptor for IgE in in vitro-generated dendritic cells.J Allergy Clin Immunol. 2000; 105: 150-156https://doi.org/10.1016/S0091-6749(00)90190-8Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). For Mo-derived CD1a+ DC it has been shown recently that intracellular α-chain (FcεRIα) accumulates during DC differentiation, whereas the expression of γ-chains (FcεRIγ) mandatory for surface expression, is downregulated. It is low or negative in DC from normal donors lacking surface FcεRI (FcεRI− DC). In contrast, CD1a+ DC from atopics show significant FcεRIγ expression and display surface FcεRI (FcεRI+ DC) (Novak et al., 2003Novak N. Tepel C. Koch S. Brix K. Bieber T. Kraft S. Evidence for a differential expression of the FcepsilonRIgamma chain in dendritic cells of atopic and nonatopic donors.J Clin Invest. 2003; 111: 1047-1056https://doi.org/10.1172/JCI200315932Crossref PubMed Scopus (109) Google Scholar). Recently, it has been demonstrated that transforming growth factor-β1 (TGF-β1) contributes to the generation of FcεRI bearing DC from circulating monocytes (Reich et al., 2001Reich K. Heine A. Hugo S. et al.Engagement of the Fc epsilon RI stimulates the production of IL-16 in Langerhans cell-like dendritic cells.J Immunol. 2001; 167: 6321-6329Crossref PubMed Scopus (54) Google Scholar). TGF-β1 is a multifunctional growth factor belonging to a superfamily of regulatory proteins and it has been suggested that variations of TGF-β in the peripheral blood and affected tissues influence the development and course of atopic diseases (Lee et al., 2000Lee H.J. Lee H.P. Ha S.J. Byun D.G. Kim J.W. Spontaneous expression of mRNA for IL-10, GM-CSF, TGF-beta, TGF-alpha, and IL-6 in peripheral blood mononuclear cells from atopic dermatitis.Ann Allergy Asthma Immunol. 2000; 84: 553-558Abstract Full Text PDF PubMed Scopus (33) Google Scholar;Arkwright et al., 2001Arkwright P.D. Chase J.M. Babbage S. Pravica V. David T.J. Hutchinson I.V. Atopic dermatitis is associated with a low-producer transforming growth factor beta(1) cytokine genotype.J Allergy Clin Immunol. 2001; 108: 281-284https://doi.org/10.1067/mai.2001.117259Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Furthermore, genetic studies aiming to detect atopy-related single nucleotide polymorphisms of the TGF-β1 gene have suggested this cytokine as a putative candidate gene for atopic conditions since they could encode for low-producer individuals (Arkwright et al., 2001Arkwright P.D. Chase J.M. Babbage S. Pravica V. David T.J. Hutchinson I.V. Atopic dermatitis is associated with a low-producer transforming growth factor beta(1) cytokine genotype.J Allergy Clin Immunol. 2001; 108: 281-284https://doi.org/10.1067/mai.2001.117259Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar;Pulleyn et al., 2001Pulleyn L.J. Newton R. Adcock I.M. Barnes P.J. TGFbeta1 allele association with asthma severity.Hum Genet. 2001; 109: 623-627https://doi.org/10.1007/s00439-001-0617-yCrossref PubMed Scopus (165) Google Scholar). These observations prompted us to investigate the putative role of TGF-β1 in the regulation of FcεRI on CD34-derived CD1a+ DC generated in vitro from CD34+ stem cells and we evaluated their functional characteristics. In a first attempt, we evaluated the effect of different concentrations of TGF-β1 on CD34+ stem cell cultures. By this, we could show that addition of TGF-β1 both at low (TGF-β1low) and high (TGF-β1high) concentrations significantly increased the generation of CD34-derived CD1a+ DC compared to control cultures (CON) without any TGF-β1 supplementation (CON: 23.90%±SEM 2.02, TGF-β1low: 56.24%±SEM (2.81 and TGF-β1high: 63.4%±SEM 3.85). It has been shown previously that a high TGF-β1 level sustains hematopoetic immaturity, whereas neutralization of endogenous and autocrine TGF-β1 sustains undifferentiated cells (Ottmann and Pelus, 1988Ottmann O.G. Pelus L.M. Differential proliferative effects of transforming growth factor-beta on human hematopoietic progenitor cells.J Immunol. 1988; 140: 2661-2665PubMed Google Scholar;Fortunel et al., 2000Fortunel N. Hatzfeld J. Aoustin L. et al.Specific dose-response effects of TGF-beta1 on developmentally distinct hematopoietic stem/progenitor cells from human umbilical cord blood.Hematol J. 2000; 1: 126-135https://doi.org/10.1038/sj.thj.6200021Crossref PubMed Scopus (27) Google Scholar). Moreover, culture condition under TGF-β1high did not only decelerate cell proliferation as indicated by lower total cell number in our system (Figure 1a) but also decelerated the differentiation of CD34+ stem cells. In contrast to CD34-derived CD1a+ DC generated under control conditions, CD34+ stem cells were still detectable in cultures with TGF-β1low and TGF-β1high after 9 d (Figure 1b). From these first series of experiments, we conclude that increasing amounts of TGF-β1 enhance the generation of CD34-derived CD1a+ DC but decelerate cell proliferation and sustain cell immaturity. To investigate the regulation of the FcεRI expression of CD34-derived CD1a+ DC during their in vitro differentiation, cells were subjected to detailed flow cytometric analyses. FcεRI appeared on the cell surface of CD34-derived CD1a+ DC under all three investigated conditions and peaked around day 5. The receptor expression stabilized at high level under control condition and TGF-β1low. In contrast, FcεRI was rapidly downregulated on CD34-derived CD1a+ DC generated with TGF-β1high and on day 9 only few cells bearing FcεRI could be detected (Figure 2a). In order to create an atopy-like microenviroment, CD34-derived CD1a+ DC were generated in the presence of IgE since it has been shown that its binding to FcεRIα may contribute to the upregulation of the receptor on APC (Reischl et al., 1996Reischl I.G. Corvaia N. Effenberger F. Wolff-Winiski B. Kromer E. Mudde G.C. Function and regulation of Fc epsilon RI expression on monocytes from non-atopic donors.Clin Exp Allergy. 1996; 26: 630-641Crossref PubMed Scopus (40) Google Scholar;Borkowski et al., 2001Borkowski T.A. Jouvin M.H. Lin S.Y. Kinet J.P. Minimal requirements for IgE-mediated regulation of surface Fc epsilon RI.J Immunol. 2001; 167: 1290-1296Crossref PubMed Scopus (136) Google Scholar). These culture conditions lead to a further significant increase of the FcεRI expression of CD34-derived CD1a+ DC (Figure 2b). Thus, FcεRI is constitutively expressed on the surface of CD34-derived CD1a+ DC during their cell differentiation from CD34+ stem cells, but this expression is subjected to further regulatory effects depending on the presence of TGF-β1 and IgE in the microenvironment. FcεRI on APC differs from the tetrameric FcεRI found on the effector cells of anaphylactic reactions by lacking the β-chain and consisting only of an IgE-binding α-chain and two disulfide linked γ-chain (Turner and Kinet, 1999Turner H. Kinet J.P. Signalling through the high-affinity IgE receptor Fc epsilonRI.Nature. 1999; 402: B24-B30https://doi.org/10.1038/35037021Crossref PubMed Scopus (620) Google Scholar). In addition, a high amount of FcεRIγ, preferently found on APC of atopic individuals, is mandatory for the FcεRI surface expression (Maurer et al., 1994Maurer D. Fiebiger E. Reininger B. et al.Expression of functional high affinity immunoglobulin e receptors (fc epsilon RI) on monocytes of atopic individuals.J Exp Med. 1994; 179: 745-750https://doi.org/10.1084/jem.179.2.745Crossref PubMed Scopus (327) Google Scholar;Maurer et al., 1996Maurer D. Fiebiger S. Ebner C. et al.Peripheral blood dendritic cells express Fc epsilon RI as a complex composed of Fc epsilon RI alpha- and Fc epsilon RI gamma- chains and can use this receptor for IgE-mediated allergen presentation.J Immunol. 1996; 157: 607-616PubMed Google Scholar). Thus, to arrive at the exact structure of FcεRI on CD34-derived CD1a+ DC and the protein level of the respective FcεRIα and FcεRIγ chains on CD34-derived CD1a+ DC, we performed extracellular and intracellular flow cytometric analyses in combination to semiquantitative PCR experiments. The latter showed comparable amounts of FcεRIα and FcεRIγ transcripts under all three cell culture conditions used (Figure 3a), whereas FcεRI β-chain remained undetectable (data not shown). Importantly, transcripts of tryptase as a control for contaminating mast cells or basophils could not be detected (data not shown). Since CD34-derived CD1a+ DC were generated in FCS, which served as a source of TGF-β1, we determined the level of endogenous TGF-β1 in the cell culture supernatant of control condition on day 9. As expected, an average of 0.03 ng per mL (SEM±0.01, n=6) TGF-β1 could be detected. This corresponds to the amount of TGF-β1 from FCS supplementation of culture medium and excludes and TGF-β1 producing source such as platelets in our cell culture system. Complete neutralization of endogenous TGF-β leads to rapid downregulation of the FcεRI surface expression on day 9. Interestingly, both receptor subunits, intracellular FcεRIα and FcεRIγ were hardly detectable and only trackable in decreased amounts after TGF-β neutralization (Figure 3b). Furthermore, control experiments revealed that supplementation of irrelevant IgG1 antibody did not alter surface expression of FcεRI (data not shown). CD34-derived CD1a+ DC generated under control conditions displayed a moderate FcεRI surface expression, whereas the highest FcεRI surface expression and intracellular FcεRI pool of the α-chain was detectable on CD34-derived CD1a+ DC generated with TGF-β1low (Figure 3c). Although the intracellular pool of FcεRIγ was elevated in CD34-derived CD1a+ DC cultured under control conditions and TGF-β1low, only low levels of FcεRIγ were detectable in CD34-derived CD1a+ DC generated in TGF-β1high (Figure 3c). Based on these cumulative data, we can conclude that the presence of TGF-β1 in the microenviroment surrounding CD34-derived CD1a+ DC is a crucial factor in the control of surface expression of FcεRI on CD34-derived CD1a+ DC. Several types of CD34-derived CD1a+ DC have been identified in the human immune system whereas TGF-β1 is critical for the generation of LC in vitro (Strobl et al., 1996Strobl H. Riedl E. Scheinecker C. et al.TGF-beta 1 promotes in vitro development of dendritic cells from CD34+ hemopoietic progenitors.J Immunol. 1996; 157: 1499-1507PubMed Google Scholar). Thus we investigated the amount of the CD1a+ LC subset under different culture conditions detected by the expression of Langerin, a lectin receptor specific for LC (Valladeau et al., 2000Valladeau J. Ravel O. Dezutter-Dambuyant C. et al.Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules.Immunity. 2000; 12: 71-81https://doi.org/10.1016/S1074-7613(00)80160-0Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar). Whereas under control conditions, we were not able to detect LC, these cells could be generated in TGF-β1low. The highest number of LC among CD34-derived CD1a+ DC could be detected under TGF-β1high. Interestingly, under TGF-β1low a remarkable amount of LC were FcεRI+ (Figure 4). Thus, we can conclude that TGF-β1 at defined concentrations (TGF-β1low) leads to the generation of FcεRI+ LC. To investigate the nature of DC generated under control condition, we performed morphological and detailed phenotypical analyses in comparison to CD34-derived CD1a+ DC generated under TGF-β1 supplementation. As reported earlier, addition of TGF-β1 leads to the formation of clusters (Riedl et al., 2000bRiedl E. Stockl J. Majdic O. Scheinecker C. Rappersberger K. Knapp W. Strobl H. Functional involvement of E-cadherin in TGF-beta 1-induced cell cluster formation of in vitro developing human Langerhans-type dendritic cells.J Immunol. 2000; 165: 1381-1386Crossref PubMed Scopus (55) Google Scholar) whereas a concomitant increase of E-cadherin—a homophilic adhesion molecule involved in the cluster formation of LC (Riedl et al., 2000aRiedl E. Stockl J. Majdic O. Scheinecker C. Knapp W. Strobl H. Ligation of E-cadherin on in vitro-generated immature Langerhans-type dendritic cells inhibits their maturation.Blood. 2000; 96: 4276-4284Crossref PubMed Google Scholar)—could be observed (Figure 5a). Although we could not find a significant difference in expression of DC-SIGN/CD209, we could detect on CD34-derived CD1a+ DC generated under control condition an increased expression of CD11b and mannose receptor (CD206) that are not only expressed by macrophages but also related to DC of non-LC type and are known to be expressed on so-called inflammatory dendritic epidermal cells (IDEC) that are usually found in lesional sites of the skin of atopic eczema patients (Sallusto et al., 1995Sallusto F. Cella M. Danieli C. Lanzavecchia A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: Downregulation by cytokines and bacterial products.J Exp Med. 1995; 182: 389-400https://doi.org/10.1084/jem.182.2.389Crossref PubMed Scopus (2180) Google Scholar;Jaksits et al., 1999Jaksits S. Kriehuber E. Charbonnier A.S. Rappersberger K. Stingl G. Maurer D. CD34+ cell-derived CD14+ precursor cells develop into Langerhans cells in a TGF-beta 1-dependent manner.J Immunol. 1999; 163: 4869-4877PubMed Google Scholar;Wollenberg et al., 2002Wollenberg A. Mommaas M. Oppel T. Schottdorf E.M. Gunther S. Moderer M. Expression and function of the mannose receptor CD206 on epidermal dendritic cells in inflammatory skin diseases.J Invest Dermatol. 2002; 118: 327-334https://doi.org/10.1046/j.0022-202x.2001.01665.xCrossref PubMed Scopus (174) Google Scholar) (Figure 5). In addition CD14 could be detected in approximately equal amounts on CD1a positive cells under all conditions (Figure 5a). Furthermore the addition of exogenous TGF-β1 augmented the expression of major histocompatibility complex (MHC) class I and II complexes and costimulatory molecules, but did not result in the final maturation of CD34-derived CD1a+ DC indicated by lack of CD83, a marker for mature DC (Figure 5b) (Zhou and Tedder, 1996Zhou L.J. Tedder T.F. CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells.Proc Natl Acad Sci USA. 1996; 93: 2588-2592https://doi.org/10.1073/pnas.93.6.2588Crossref PubMed Scopus (957) Google Scholar). To investigate the influence of TGF-β1 on the functional properties of FcεRI+CD34-derived CD1a+ DC, we tested their stimulatory activity in an allogeneic system. As shown in Figure 6, the addition of TGF-β1 leads to generation of CD34-derived CD1a+ DC, which display a significant increased allogeneic stimulatory activity. TGF-β1 is a pleiotropic regulator of the hematopoesis, which displays a growth inhibitory effect on early progenitor cells and influences distinctly the differentiation of CD34-derived CD1a+ DC (Fortunel et al., 2000Fortunel N. Hatzfeld J. Aoustin L. et al.Specific dose-response effects of TGF-beta1 on developmentally distinct hematopoietic stem/progenitor cells from human umbilical cord blood.Hematol J. 2000; 1: 126-135https://doi.org/10.1038/sj.thj.6200021Crossref PubMed Scopus (27) Google Scholar). In this study, we investigated the involvement of TGF-β1 in the regulation of the FcεRI α- and γ-chains and the differentiation of FcεRI bearing subsets of CD34-derived CD1a+ DC. We uncovered that especially the intracellular FcεRIγ pool, which is mandatory for sufficient FcεRI surface expression, is distinctly adjusted by the amount of TGF-β1, whereas the regulation of FcεRIγ subunits occur at the protein but not at the mRNA level suggesting that TGF-β is involved in the regulation of the γ-chain at a post-transcriptional point. Another group reported similar findings of γ-chain regulation in Mo-derived macrophage where infection of the cells with HIV lead to downregulation of γ-chain on protein level but did not alter the mRNA expression (Kedzierska et al., 2002Kedzierska K. Ellery P. Mak J. Lewin S.R. Crowe S.M. Jaworowski A. HIV-1 down-modulates gamma signaling chain of Fc gamma R in human macrophages: A possible mechanism for inhibition of phagocytosis.J Immunol. 2002; 168: 2895-2903Crossref PubMed Scopus (75) Google Scholar). Just recently, another group reported that TGF-β1 suppressed expression of Fcγ–receptors on human myeloid cells by downregulating the γ-chain, which is identical to FcεRIγ (Ra et al., 1989Ra C. Jouvin M.H. Blank U. Kinet J.P. A macrophage Fc gamma receptor and the mast cell receptor for IgE share an identical subunit.Nature. 1989; 341: 752-754https://doi.org/10.1038/341752a0Crossref PubMed Scopus (220) Google Scholar; Tridandapani et al, 2003). At early differentiation stages, FcεRI is constitutively expressed on the cell surface of the differentiating CD34-derived CD1a+ DC. Later on, low or moderate TGF-β1 levels facilitate the differentiation of FcεRI bearing CD34-derived CD1a+ DC subtypes such as Langerin+ LC and Langerin− CD34-derived CD1a+ DC. In contrast, the addition of TGF-β1 at high doses and the total elimination of TGF-β from the surrounding micromilieu prevent the generation of FcεRI+ CD34-derived CD1a+ DC subtypes during further differentiation and accelerate the differentiation of FcεRI− CD34-derived CD1a+ DC such as LC and Langerin− CD34-derived CD1a+ DC. TGF-β1 seems to play a major role in the regulation of FcεRI since the generation of the different FcεRI+ DC subtypes in combination with a distinct intracellular FcεRIα and FcεRIγ pool were influenced by addition of exogenous TGF-β1. As we used a TGF-β blocking antibody which was directed against TGF-β1, β2, and β3, we cannot exclude that downregulation of FcεRI results from an cooperative effect of all TGF-β isoforms. Even though increasing evidence arises that TGF-β1 plays a pivotal role in atopic diseases, its influence on the expression of the high-affinity receptor for IgE on human APC has not been investigated in detail before. In the past, studies have shown that atopic diseases such as atopic dermatitis go along with a low TGF-β1 producing phenotype (Lee et al., 2000Lee H.J. Lee H.P. Ha S.J. Byun D.G. Kim J.W. Spontaneous expression of mRNA for IL-10, GM-CSF, TGF-beta, TGF-alpha, and IL-6 in peripheral blood mononuclear cells from atopic dermatitis.Ann Allergy Asthma Immunol. 2000; 84: 553-558Abstract Full Text PDF PubMed Scopus (33) Google Scholar;Arkwright et al., 2001Arkwright P.D. Chase J.M. Babbage S. Pravica V. David T.J. Hutchinson I.V. Atopic dermatitis is associated with a low-producer transforming growth factor beta(1) cytokine genotype.J Allergy Clin Immunol. 2001; 108: 281-284https://doi.org/10.1067/mai.2001.117259Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). These findings are largely compatible with recent data, which predict that in some instances defects in the TGF-β1 production contribute to human diseases such as allergic hypersensitivity disorders (Sugai et al., 2003Sugai M. Gonda H. Kusunoki T. Katakai T. Yokota Y. Shimizu A. Essential role of Id2 in negative regulation of IgE class switching.Nat Immunol. 2003; 4: 25-30https://doi.org/10.1038/ni874Crossref PubMed Scopus (110) Google Scholar). These are characterized by an excessive IgE serum level in combination with an enhanced FcεRI surface expression on DC in the peripheral blood and tissues. Further on, it has been shown that TGF-β is largely involved in the regulation of the IgE synthesis itself and displays a downregulatory function on IgE serum levels (Wu et al., 1992Wu C.Y. Brinkmann V. Cox D. Heusser C. Delespesse G. Modulation of human IgE synthesis by transforming growth factor-beta.Clin Immunol Immunopathol. 1992; 62: 277-284https://doi.org/10.1016/0090-1229(92)90103-UCrossref PubMed Scopus (24) Google Scholar;Sugai et al., 2003Sugai M. Gonda H. Kusunoki T. Katakai T. Yokota Y. Shimizu A. Essential role of Id2 in negative regulation of IgE class switching.Nat Immunol. 2003; 4: 25-30https://doi.org/10.1038/ni874Crossref PubMed Scopus (110) Google Scholar). In healthy, non-atopic individuals, high TGF-β serum levels prevail. The epidermal skin lesions of atopic dermatitis are characterized by high numbers of FcεRI-bearing LC and another FcεRI bearing DC subtype, the so-called IDEC. In contrast in the skin of healthy, non-atopic individuals IDEC are completely absent and FcεRI− LC attend in the epidermis (Wollenberg et al., 1995Wollenberg A. Wen S. Bieber T. Langerhans cell phenotyping: A new tool for differential diagnosis of inflammatory skin diseases [letter].Lancet. 1995; 346: 1626-1627https://doi.org/10.1016/S0140-6736(95)91958-9Abstract PubMed Scopus (70) Google Scholar). In view of the data presented here, low or moderate TGF-β1 producing phenotype which are a pathognomonic feature of atopic individuals could promote the development of FcεRI+ DC subtypes in vivo. Since FcεRI appears on the cell surface of CD34-derived CD1a+ DC during differentiation, IgE as a receptor stabilizing mechanism via FcεRIα binding (Borkowski et al., 2001Borkowski T.A. Jouvin M.H. Lin S.Y. Kinet J.P. Minimal requirements for IgE-mediated regulation of surface Fc epsilon RI.J Immunol. 2001; 167: 1290-1296Crossref PubMed Scopus (136) Google Scholar) might further contribute to the high FcεRI expression on DC in atopic individuals, whereas in an non-atopic enviroment regular TGF-β levels could inhibit FcεRI surface expression on DC. Together, these components might trigger the generation of the pathophysiologically relevant FcεRI+ DC subtypes, which might perpetuate the course of pro-inflammatory and allergic processes of atopic diseases by their high antigen-presenting and stimulatory capacities in vivo. Whether the wide margin of fluctuation of active TGF-β1 levels in the human cord blood might bias or prevent selectively the development of these potent players in the atopic game in vivo and, therefore, serve as a predictive factor for the generation of atopic diseases later in life, remains to be elucidated. Phycoerythrin (PE)-labeled T6RD1 (IgG1, Beckman Coulter, Krefeld, Germany), unlabeled IOT6a (IgG1, Beckman Coulter) and Cy-Chrome-labeled HI149 (IgG1, Beckton Dickinson (BD), Heidelberg, Germany) recognize CD1a. FcεRI was detected by mAb 22E7 (IgG1) directed against the α-chain not interfering with the IgE binding site; mAb 4D8 (IgG2b) is directed against the γ-chain. These antibodies were prepared and characterized as described elsewhere (Riske et al., 1991Riske F. Hakimi J. Mallamaci M. et al.High affinity human IgE receptor (Fc epsilon RI). Analysis of functional domains of the alpha-subunit with monoclonal antibodies.J Biol Chem. 1991; 266: 11245-11251Abstract Full Text PDF PubMed Google Scholar;Schoneich et al., 1992Schoneich J.T. Wilkinson V.L. Kado-Fong H. Presky D.H. Kochan J.P. Association of the human Fc epsilon RI gamma subunit with novel cell surface polypeptides.J Immunol. 1992; 148: 2181-2185PubMed Google Scholar); mAb IOT2b (IgG1) directed against HLA-DR, mAb M5E2 (IgG2a) directed against CD14, mAb BEAR-1 (IgG1) detecting CD11b and mAb HB15a detecting CD83 (IgG2b) were purchased from (Immunotech, Marseille, France). MAb DCN46 (IgG2b) against DC-SIGN/CD209, mAb 19 (IgG1) detecting CD206 and mAb B70/B7-2 (IgG1) directed against CD86 and mAb 5C3 (IgG1) directed against CD40 (IgG1) were from BD. Mab. BB-1/B7 (IgG1) reacting with CD80 and PE-labeled 8G12 (IgG1) directed against CD34 were from BD, purified and PE-labeled mAb DCGM4 (IgG1) recognizes LC-specific Birbeck Granules (BG)-associated lectin Langerin/CD207 (Immunotech). MAb 67A4 (IgG1) detecting E-cadherin was purchased from Beckman-Coulter. MOPC-21 (IgG1, Sigma, Deisenhofen, Germany), mAb 1D11 is directed against TGF-β1, -β2, -β3 (IgG1, R&D Systems, Minneapolis, Minnesota). MOPC-21 (IgG1), UPC10 (IgG2a), MOPC-141 (IgG2b) all from Sigma and IgG1RD1 (Beckman-Coulter) were used as appropriate isotype controls. Fluorescein-isothiocyanate (FITC)-conjugated goat anti-mouse (GaM/FITC) antibody was from Jackson Laboratories (West Grove, Pennsylvania). Sheep anti-mouse coated magnetic beads (M-280) were obtained from Dynal (Oslo, Norway). Normal mouse serum for blocking purposes, 7-aminoactinomycin-D (7AAD) and 2-mercaptoethanol were from Sigma. Human myeloma IgE (PS) was obtained from Calbiochem (Bad Soden, Germany). Heat-inactivated fetal calf serum (FCS), antibiotics/antimycotics and RPMI-1640 with Glutamax were all from Invitrogen (Carlsbad, California). Granulocyte macrophage colony stimulating factor (GM-CSF) was purchased from Novartis Pharma (Nürnberg, Germany), tumor necrosis factor (TNF-β, stem cell factor (SCF), Flt-3-ligand (FL3) and TGF-β1 were purchased from R&D Systems. Cord blood was obtained from vaginal or cesarean deliveries after informed written consent from patients according to local Medical Ethics Committee of the University of Bonn, Germany and the study was performed according to Declaration of Helsinki Guidelines. After the blood was diluted three times with RPMI-1640 (Invitrogen), 25 mL of suspended cells was overlayed on 15 mL of Lymphoprep (Nycomed, Oslo, Norway). peripheral blood mononuclear cells (PBMC) were isolated as interface cells after density gradient centrifugation (20 min at 900 ×g at room temperature). To reduce residual platelets, the recovered cells were washed twice in PBS and recovered in PBS supplemented with 1% BSA and 5 mmol EDTA. Isolation of CD34+ cells was performed using magnetic microbeads coupled to anti-CD34 antibody and the AUTOMACS system (Milteny Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol. The enrichment of CD34+ cells was determined with an PE-conjugated anti-CD34 mAb and was more than 90% CD34+ cells. 4 × 104 purified CD34+ cells were cultured in 24-well plates (Corning, Acton, Massachusetts) with 0.5 mL RPMI-1640 with Glutamax (Invitrogen) supplemented with 10% heat inactivated FCS (Invitrogen), 1% antibiotics/antimycotics Invitrogen) and 5 μM ME (Sigma). Since it has been shown that combination of GM-CSF, TNF-α, SCF, FL3, and TGF-β1 give rise to a great number of CD34-derived CD1a+ DC (Strobl et al., 1997Strobl H. Bello-Fernandez C. Riedl E. Pickl W.F. Majdic O. Lyman S.D. Knapp W. flt3 ligand in cooperation with transforming growth factor-beta1 potentiates in vitro development of Langerhans-type dendritic cells and allows single-cell dendritic cell cluster formation under serum-free conditions.Blood. 1997; 90: 1425-1434PubMed Google Scholar), we used GM-CSF (300 IU per mL), TNF-β (50 IU per mL), SCF (10 ng per mL), and FL3 (10 ng per mL) in FCS supplemented medium as control culture conditions. We performed ELISA to analyze the amount of TGF-β1 in the FCS charges used for medium supplementation. We detected an average TGF-β1 concentration of 0.39 ng per mL (SEM±0.09; n=3). Since we used 10% FCS supplemented medium we expected an endogenous TGF-β1 concentration of 0.04 ng per mL. Hereafter, the baseline condition will be referred to as control. According to TGF-β1 levels found in cord blood (0.89 ng per mL, SEM±0.08, n=16) and to a previous study (Strobl et al., 1996Strobl H. Riedl E. Scheinecker C. et al.TGF-beta 1 promotes in vitro development of dendritic cells from CD34+ hemopoietic progenitors.J Immunol. 1996; 157: 1499-1507PubMed Google Scholar), we defined low and high exogenous TGF-β1 culture conditions as follows: low TGF-β1 (TGF-β1low) with 0.5 ng per mL and high TGF-β1 (TGF-β1high) with 10 ng per mL which were added to control conditions. The cells obtained will be refered to as CD34-derived CD1a+ DC. Generation and culture of CD34-derived CD1a+ DC were maintained for 9 d for kinetic experiments. One-half of the medium was replaced with fresh cytokines after 4 and 8 d. To mimic an atopic environment CD34+ cells were cultured as described above in addition to 1 μg per mL human myeloma IgE (every other days). Photographs were taken with a DMCRB microscope (Leica, Bensheim, Germany) using an HV-C2OA digital camera (Hitachi, Duesseldorf, Germany) and Diskus software (Hilgers, Koenigswinter, Germany). For RNA extraction, CD1a+ DC were enriched from the culture conditions using an anti-CD1a mAb bound to magnetic beads according to the manufacturer's protocol. Briefly, Dynabeads M-280 precoated with sheep anti-mouse mAb were incubated with anti-CD1a mAb IOT6. After washing procedures, DC cell suspensions were added to the bead suspensions and incubated for 1 h at 4°C. CD1a+ cells bound to the beads were then purified by four rounds of washing procedures with the magnet, yielding purity greater than 98% as controlled by light microscopy. Double-staining experiments with saponin or digitonin for the detection of surface or intracellular distribution of FcεRI subunits were performed as described in detail (Kraft et al., 1998Kraft S. Wessendorf J.H. Hanau D. Bieber T. Regulation of the high affinity receptor for IgE on human epidermal Langerhans cells.J Immunol. 1998; 161: 1000-1006PubMed Google Scholar). For triple-staining experiments cells were incubated with Cy-chrome-labeled CD1a and PE-labeled Langerin after permeabilization. Saponin permeabilization was used for detection of intracellular FcεRIα, whereas digitonin permeabilization was used for detection of FcεRIγ expression. Cells were acquired using a FACSCalibur flow cytometer (BD) as described in detail elsewhere (Novak et al., 2001Novak N. Bieber T. Katoh N. Engagement of FcepsilonRI on human monocytes induces the production of IL-10 and prevents their differentiation in dendritic cells.J Immunol. 2001; 167: 797-804Crossref PubMed Scopus (105) Google Scholar). For quantitative evaluation, the CD1a+ population was gated manually and the percentage of FcεRIα+ or FcεRIγ+ cells was determined using Lysis II software (BD). Immunolabeling for phenotyping was performed as reported (Wollenberg et al., 1999Wollenberg A. Wen S. Bieber T. Phenotyping of epidermal dendritic cells: Clinical applications of a flow cytometric micromethod.Cytometry. 1999; 37: 147-155Crossref PubMed Scopus (90) Google Scholar). As a control, cells were stained with corresponding isotype-matched control immunoglobulin. All incubations and washes were performed at 4°C. Results are expressed as percent of positive cells. TGF-β1 levels in cord blood plasma and culture supernatants were determined by ELISA according to the manufacturer's protocol (Quantikine, R&D Systems) and as described in detail (Novak et al., 2001Novak N. Bieber T. Katoh N. Engagement of FcepsilonRI on human monocytes induces the production of IL-10 and prevents their differentiation in dendritic cells.J Immunol. 2001; 167: 797-804Crossref PubMed Scopus (105) Google Scholar). Total RNA was extracted after 9 d of culture from highly purified CD34-derived CD1a+ DC using Trizol (Life Technologies, Eggenstein, Germany) following the manufacturer's instructions. RT reactions were performed using 1 μg of total RNA. Denaturation at 94°C for 40 s was followed by annealing of the primers at 55°C for 30s and extension at 72°C for 30s. A final extension phase of 5 min was added. Specific primer sequences for each gene were as follows: human β-actin: sense, 5′-GAG CGG GAA ATC GTG CGT GAC ATT-3′; antisense, 5′-GAT GGA GTT GAA GGT AGT TTC GTG-3′, yielding a fragment of 240 bp; human FcεRIγ: sense, 5′-CTG TTC TTC GCT CCA GAT GGC GT -3′; antisense, 5′-TAC AGT AAT GTT GAG GGG CTC AG -3′ (fragment of 536 bp), human FcεRIγsense, 5′-CCA GCA GTG GTC TTG CTC TTA C-3′ and antisense: 5′-GCA TGC AGG CAT ATG TGA TGC C-3′ (fragment of 338bp), human FcεRIβ sense, 5′-GGA CAC AGA AAG TAA TAG GAG AG and antisense: 5′-GAT CAG GAT GGT AAT TCC CGT T (fragment of 446 bp) and human tryptase sense, 5′-CTC CCT CAT CCA CCC CCA GT and antisense: 5′-GGA TCC AGT CCA AGT AGT AG (fragment of 616 bp). Amplification was performed on a Perkin-Elmer Gene Amp PCR System 9600 thermocycler (Applied Biosystems, Weiterstadt, FRG). The PCR cycle numbers for the amplification of the respective cDNAs were 25 for β-actin and 30 for FcεRIα and FcεRIγ. Specific PCR fragments were separated on a 1% agarose gel and visualized using ethidium bromide staining. The PCR products were evaluated semiquantitatively by comparing the ratio of the specific products versus the β-actin band by digital image analysis using the WinCam system (Cybertech, Berlin, Germany). Proliferation assays were performed in a total volume of 200 μL in 96-well round-bottom plastic culture plates using allogeneic T cells as responder cells. Allogeneic T cells were isolated from PBMC of healthy volunteers using a nylon-wool column (>85% purity as assessed by anti-CD3 staining). DC were cultured as described above until day 9 of culture. In allogeneic assays, triplicates of DC containing 100, 200, or 1000 viable CD1a+ DC/well were incubated with 100,000 viable allogeneic T cells at 37°C for 3 d. Proliferative response was then measured by addition of 1 μCi 3H-thymidine incorporation for 12 h. The incorporated radioactivity was measured in a liquid scintillation counter (1450 MicroBeta Trilux, Wallac). Relative stimulation indices (rSI) were calculated as follows: rSI=(c.p.m. (DC+T cells)-c.p.m. (T cells)/cpm (T cells). For statistical evaluation of significances, the Wilcoxon test was performed. Correlations were calculated by Pearson's linear regression analysis. These tests were realized using the SPSS 10.0 software (SPSS, Chicago, lllinois). Results are shown as arithmetic mean±standard error of the mean (SEM). *, p<0.05, **, p<0.01, n.s., not significant. The studies performed were approved by the local institution. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG NO-454/1/1) and BONFOR.
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