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langerhans cells along their in vitro differentiation from CD34+ progenitor cells
s15
ESDR I JSID I ND Abstracts
0088
0085
Ex ressioo of ehemokine receptors on dendritie celWLq~ erbnns c# dif!ereot&?tioa from CD34+ pro enitor cells. Anne-So~lue ?zbarbonmer
$i$$$
StingI. Dieter Mawer,
Dh D, Depamoent oFi%rmetology,
along their in vitro
Norbert K&r her. Umverdity of Vienna iF,edlcal
Abstract not supplied
0089
0086 MATURE MTERFERON?
HUMAN
DENDRlTlC
PRODUClNG
MI
CELLS CELLS
ARE AND
EFFICIENT ARE
UNABLE
INDUCERS TO
INDUCE
OF TH2
CELLS EVEN IN THE PRESENCE OF IL-4 AND ANTI-IL-12. H. Jonuleit. E Schmitt*. K. Steinbrink. 1.3. h#jller. C Sralma. J Knop, and A. Eok Dept of Dermatol cod *Io~Ut~te of Immunol., Vowers. ofMaw, Mainz, Germany. Culture conditions for human dendritio cells (DC) have been developed by szvcrel grows Most of them oti FCScontaining conditions to generate DC in the presence of CM-CSF and IL-4
RecenUy. it was shown that these cells represent rather immatwe DC that need as addtlional stimulation lor the final dillerenlialion into mature DC It was show that ao addioonal rtimulaooe of DC-prewtwrs with the defined cytoktne-cocktail IL-VlVFIl~ + PG& reduce an irreverslblc terminal dilferenlralion into metore DC. Here we iovestigated whether the diRerentirtion-scale ol DC can inlluenae the wtceme 01 the T celt (TC) response. Therelore we used mooatore DC and matore DC after temonat maturatloo for the stimolauon of alloecoeic. naive cordblmd-denved T
cells in different DCTC-ratios. T cells were restinwlald 2-3 weeks later wth an&CD3 and eel,CD28 mAb or wtb DC derived from the same donor as in the primer) stonulation. WC repon that immeture DC predomrnantly indocz a ThO- or Thlpheootyp dependcot on the DC:TC-ratio whereas mature DC mduce a dominant Thl-phenotype medependent of the DCTC-ratio (l:2 to l:lZSO). At a high DC:TC-ratio (l:2 to l:to) immature DC cultured in FCS-cootaininr conditions induce IFN-~w/lL-4w producing T cells At DC TC-rabo lower than l 20 mwnatu~DC mduce lFN-y”d/lL-4”d producing T cells In contrast. after tcmtinal mawation of DC-precursors m the eresence of lLIm/IL6 + PGE? mature DC induce onlv IF&‘* oroducinr T cells indeocndent
bftk
DC:Ttket~o cl:2 to 1:1250) and are not able to i&oce Ii-4 s)-othests;o naiw ello~encic T cells Furthermore. even in the presence of IL-4 during primal) sumulation mature DC failed to Induce IL-4 prodoct~on m T cells. although IFN-7 qntbesis io these cultures was reduced Neutralization of IL-12 wth mAb during prima5 stimulation amin inhlbw IFN-Y ~~nthens of T
cells st~mulatcd wth mature DC. but comoletlv failed to re& IL-4 nnthesis m ihe T ~~11s Thus. terminally matured DC are potent induce;s of Thl cells. bet inca~ble of inducmg Th2 cells even in the presence of IL-4 and neutraliation &IL-12
HAPTOGLOBIN IS A NATURAL HOMEOSTATIC FACTOR THAT PREVENTS EPIDERMAI. LANGERHANS CELLS FROM ACTIVATING NAIVE, SYNGENEIC T CELLS IN SITU. Yone Xie. Zhane. Yanhua Li. Matthew J. Stiller. Albert Wane. and 1. Wavne Streilein. Schepens Eye Research Institute, Boston Biomedical Research Institute and Harvard Department of Dermatology, Boston, MA, USA Langerhans cells (LC) in the skin can capture, process and present exogenous antigens in situ to previously primed, but uot to unprimed (naive), T cells. Once antigen-bearing LC leave the skin during sensitization they undergo a functional transformation in which enhanced surface expression of class II MHC molecules and potent co-stimulatoty signals enable the cells to present antigen to naive T cells in draining lymph nodes. It has been postulated that T cell-dependent skin mflammatory diseases may result if LC acquire these novel properties inappropriately within the epidermis. We have recently reported that both human and murine sera contain a factor that prevents freshly procured epidermal LC from acquiring these novel properties in vitro. and we have proposed that this factor acts in vivo to constrain epidemml LC from adopting potent co-stimulation properties in situ. We have now identified the relevant serum factor as haptoglobin (or its alpha-l chain), based on the following results: (I)SDS PAGE, amino acid sequencing, and mass spectrometric analyses of the inhibitory factor purified by HPLC from normal human serum revealed a molecule completely homologous to haptoglobin alpha-l chain: (2) pure human baptoglobin, but not serum depleted of haptoglobin (antibody affinity chromatography), Inhibited GMCSF-treated fresh LC from acquiring the capacity to activate syngenex T cells in vitro; (3) abundant haptoglobin (fluorescence mtcroscopy) was found in cytoplasmtc vesicles of fresh LC. We conclude that haptoglobin, an acute-phase response serum protein, is a systemic factor that constrains epidermal LC to display antigen presenting properties suitable to the cutaneous microenvironment.
Oiang
0087
0090
ADHERENS JUNCTION-LIKE CONTACTS OF SKIN-DERIVED DENDRITIC CELLS AND KERATINOCYTES IN VITRO. z J&oh. M J m* and ML w. Dermatology and *Experimental Immunology Branches, NC& Bethesda, MD Adherens iunctions are adhesion stnxtures in which intercellular attachment is mediated by cadherins that are linked to the actin cytoskeleton via cytoplasmic catenins. Ecadherin (E-cad) constitutes the major adhesion molecule of adherens junctions in keratinocytes and mediates binding of Langerhans cells (LC) to keratinccytes (KC) in vitro. To further characterize E-cad-mediated adhesion involving LC, we utilized rhe recently described murine fetal skin-derived dendritic &I (FSDDC) model (J. Immunol. 159:2693, 1997). Confocal microscopy of FSDDC aggregates in which adhesion is &ad-mediated revealed colocalization of E-cad and catcoins ($-, ~-cat&n. p~l2@~) to areas of c&c&l contact. Coimmunoprecipitation studies indicated that, iu FSDDC, E-cad associated with a-, B-, and ~catenin and pp120c*s. Transmission electron microscopy (TEM) of FSDDC aggregates revealed adherens junction-like c&Iceil contacts comprised of iotmcellular plaques of incrased electron density with electmn dense mat&al in the intercellular space. Postembedding immunelectron microscopy suggested preferential accumulation Of p-cat&n in these c&cell junctions. To determine if similar structures accounted for the adhesion of LC-like DC and KC. FSDDC agg~??gates were dissociated with EDTA and single cells were cocuhured with primary mwine KC. After 18 h in 1 mM Ca” containing-media. cells were fved and processed for ‘IEM or stained for MHC class II and E-cad. TEM analysis showed adherens junction-like contacts between KC and DC. Confocal microscopy demonstrated foal accumulations of E-cad in eras of contact between KC and LC-like immature FSDDC (high intracellular, low surface MHC class II), but not between KC and mature DC (low intracellular, high surface MHC class II). We suggest that E-cad in LC is localized in adherens junction-like st~ctures in situ, and that the loss of ability to form or maintain similar SO’octor~ after LC activation is responsible for the attenuation of LC-KC adhesion that allows LC to emigrate from epidermis.
NF&/REL PROTEIN EXPRESSION AND ACTIVITY IN A MODEL OF DENDRITIC CELL,(DC) ,ACITVATION AND MATURATION. A. Kikuchif, G.Franzoso#. *Dermatology Branch, NC1 and XLaboratoty of Immunoregulation, NIAID, Bethesda, MD USA NF&Rel transcription factors are widely distributed in hematopoetic cells and play critical mles in the initiation and regulation of immune and inflammatory responses. Becaw NFrB/Rel proteins w activated by known DC activators (ILl/TNFo/LPS) and at least one family member (RelB) is reported to be required for development of interdigitating DC, we assessed NFrBmel protein expression and activity in an in vitro system that models DC activation and maturation. Mutie Langedams cell-like DC [immatum fetal skin-derived DC (FSDDC-I)] were expanded in primary cultures and isolated as previously described (I. Immunol. 159: 2693, 1997). In some expaiments, FSDDC-I were allowed to develop into &Is resembling mature interdigitating DC (FSDDC-M) during a several day subculture period. and in others maturation was induced by LPS (100 ng/mI). Immunofluorescence microscopy studies revealed litde if any ReIB in FSDDC-I, although RelA was detected. In contrast, RelB staining was intense in FSDDC-M and was localized to the nucleus. Quantitation of NFrSlRel proteins in cytoplasm plus nuclear FSDDC lysates (II&) by immunoblotting demonstrated a -20-fold induction of RelB during DC maturation. Similar amounts of RelB mRNA were detected in FSDDC-I and FSDDC-M by nonhero analysis, suggesting post-transcriptional regulation of RelB. Characterization of NFrB/Rel complexes by electrophoretic mobility shift assay (II+ showed that levels of active NF&/Rel hctcrodimen m FSDDC-M were 2.3-fold higher than in FSDDC-I. Almost alI NFrBiRel heterodimers in FSDDC-I contained RelA while NF&/Rel heterodimers in FSDDC-M were R&3 predominant. Finally, time course studies in which maturation was induced by LPS revealed coordinate upregulation of RelB, MHC class II and CD86. RelB may play an impatant role in DC activationlmaturation. Our in vitro model system should facilitate definitive studies of RelB metabolism and function in DC.
ESDR I JSID I ND Abstracts
0088
0085
Ex ressioo of ehemokine receptors on dendritie celWLq~ erbnns c# dif!ereot&?tioa from CD34+ pro enitor cells. Anne-So~lue ?zbarbonmer
$i$$$
StingI. Dieter Mawer,
Dh D, Depamoent oFi%rmetology,
along their in vitro
Norbert K&r her. Umverdity of Vienna iF,edlcal
Abstract not supplied
0089
0086 MATURE MTERFERON?
HUMAN
DENDRlTlC
PRODUClNG
MI
CELLS CELLS
ARE AND
EFFICIENT ARE
UNABLE
INDUCERS TO
INDUCE
OF TH2
CELLS EVEN IN THE PRESENCE OF IL-4 AND ANTI-IL-12. H. Jonuleit. E Schmitt*. K. Steinbrink. 1.3. h#jller. C Sralma. J Knop, and A. Eok Dept of Dermatol cod *Io~Ut~te of Immunol., Vowers. ofMaw, Mainz, Germany. Culture conditions for human dendritio cells (DC) have been developed by szvcrel grows Most of them oti FCScontaining conditions to generate DC in the presence of CM-CSF and IL-4
RecenUy. it was shown that these cells represent rather immatwe DC that need as addtlional stimulation lor the final dillerenlialion into mature DC It was show that ao addioonal rtimulaooe of DC-prewtwrs with the defined cytoktne-cocktail IL-VlVFIl~ + PG& reduce an irreverslblc terminal dilferenlralion into metore DC. Here we iovestigated whether the diRerentirtion-scale ol DC can inlluenae the wtceme 01 the T celt (TC) response. Therelore we used mooatore DC and matore DC after temonat maturatloo for the stimolauon of alloecoeic. naive cordblmd-denved T
cells in different DCTC-ratios. T cells were restinwlald 2-3 weeks later wth an&CD3 and eel,CD28 mAb or wtb DC derived from the same donor as in the primer) stonulation. WC repon that immeture DC predomrnantly indocz a ThO- or Thlpheootyp dependcot on the DC:TC-ratio whereas mature DC mduce a dominant Thl-phenotype medependent of the DCTC-ratio (l:2 to l:lZSO). At a high DC:TC-ratio (l:2 to l:to) immature DC cultured in FCS-cootaininr conditions induce IFN-~w/lL-4w producing T cells At DC TC-rabo lower than l 20 mwnatu~DC mduce lFN-y”d/lL-4”d producing T cells In contrast. after tcmtinal mawation of DC-precursors m the eresence of lLIm/IL6 + PGE? mature DC induce onlv IF&‘* oroducinr T cells indeocndent
bftk
DC:Ttket~o cl:2 to 1:1250) and are not able to i&oce Ii-4 s)-othests;o naiw ello~encic T cells Furthermore. even in the presence of IL-4 during primal) sumulation mature DC failed to Induce IL-4 prodoct~on m T cells. although IFN-7 qntbesis io these cultures was reduced Neutralization of IL-12 wth mAb during prima5 stimulation amin inhlbw IFN-Y ~~nthens of T
cells st~mulatcd wth mature DC. but comoletlv failed to re& IL-4 nnthesis m ihe T ~~11s Thus. terminally matured DC are potent induce;s of Thl cells. bet inca~ble of inducmg Th2 cells even in the presence of IL-4 and neutraliation &IL-12
HAPTOGLOBIN IS A NATURAL HOMEOSTATIC FACTOR THAT PREVENTS EPIDERMAI. LANGERHANS CELLS FROM ACTIVATING NAIVE, SYNGENEIC T CELLS IN SITU. Yone Xie. Zhane. Yanhua Li. Matthew J. Stiller. Albert Wane. and 1. Wavne Streilein. Schepens Eye Research Institute, Boston Biomedical Research Institute and Harvard Department of Dermatology, Boston, MA, USA Langerhans cells (LC) in the skin can capture, process and present exogenous antigens in situ to previously primed, but uot to unprimed (naive), T cells. Once antigen-bearing LC leave the skin during sensitization they undergo a functional transformation in which enhanced surface expression of class II MHC molecules and potent co-stimulatoty signals enable the cells to present antigen to naive T cells in draining lymph nodes. It has been postulated that T cell-dependent skin mflammatory diseases may result if LC acquire these novel properties inappropriately within the epidermis. We have recently reported that both human and murine sera contain a factor that prevents freshly procured epidermal LC from acquiring these novel properties in vitro. and we have proposed that this factor acts in vivo to constrain epidemml LC from adopting potent co-stimulation properties in situ. We have now identified the relevant serum factor as haptoglobin (or its alpha-l chain), based on the following results: (I)SDS PAGE, amino acid sequencing, and mass spectrometric analyses of the inhibitory factor purified by HPLC from normal human serum revealed a molecule completely homologous to haptoglobin alpha-l chain: (2) pure human baptoglobin, but not serum depleted of haptoglobin (antibody affinity chromatography), Inhibited GMCSF-treated fresh LC from acquiring the capacity to activate syngenex T cells in vitro; (3) abundant haptoglobin (fluorescence mtcroscopy) was found in cytoplasmtc vesicles of fresh LC. We conclude that haptoglobin, an acute-phase response serum protein, is a systemic factor that constrains epidermal LC to display antigen presenting properties suitable to the cutaneous microenvironment.
Oiang
0087
0090
ADHERENS JUNCTION-LIKE CONTACTS OF SKIN-DERIVED DENDRITIC CELLS AND KERATINOCYTES IN VITRO. z J&oh. M J m* and ML w. Dermatology and *Experimental Immunology Branches, NC& Bethesda, MD Adherens iunctions are adhesion stnxtures in which intercellular attachment is mediated by cadherins that are linked to the actin cytoskeleton via cytoplasmic catenins. Ecadherin (E-cad) constitutes the major adhesion molecule of adherens junctions in keratinocytes and mediates binding of Langerhans cells (LC) to keratinccytes (KC) in vitro. To further characterize E-cad-mediated adhesion involving LC, we utilized rhe recently described murine fetal skin-derived dendritic &I (FSDDC) model (J. Immunol. 159:2693, 1997). Confocal microscopy of FSDDC aggregates in which adhesion is &ad-mediated revealed colocalization of E-cad and catcoins ($-, ~-cat&n. p~l2@~) to areas of c&c&l contact. Coimmunoprecipitation studies indicated that, iu FSDDC, E-cad associated with a-, B-, and ~catenin and pp120c*s. Transmission electron microscopy (TEM) of FSDDC aggregates revealed adherens junction-like c&Iceil contacts comprised of iotmcellular plaques of incrased electron density with electmn dense mat&al in the intercellular space. Postembedding immunelectron microscopy suggested preferential accumulation Of p-cat&n in these c&cell junctions. To determine if similar structures accounted for the adhesion of LC-like DC and KC. FSDDC agg~??gates were dissociated with EDTA and single cells were cocuhured with primary mwine KC. After 18 h in 1 mM Ca” containing-media. cells were fved and processed for ‘IEM or stained for MHC class II and E-cad. TEM analysis showed adherens junction-like contacts between KC and DC. Confocal microscopy demonstrated foal accumulations of E-cad in eras of contact between KC and LC-like immature FSDDC (high intracellular, low surface MHC class II), but not between KC and mature DC (low intracellular, high surface MHC class II). We suggest that E-cad in LC is localized in adherens junction-like st~ctures in situ, and that the loss of ability to form or maintain similar SO’octor~ after LC activation is responsible for the attenuation of LC-KC adhesion that allows LC to emigrate from epidermis.
NF&/REL PROTEIN EXPRESSION AND ACTIVITY IN A MODEL OF DENDRITIC CELL,(DC) ,ACITVATION AND MATURATION. A. Kikuchif, G.Franzoso#. *Dermatology Branch, NC1 and XLaboratoty of Immunoregulation, NIAID, Bethesda, MD USA NF&Rel transcription factors are widely distributed in hematopoetic cells and play critical mles in the initiation and regulation of immune and inflammatory responses. Becaw NFrB/Rel proteins w activated by known DC activators (ILl/TNFo/LPS) and at least one family member (RelB) is reported to be required for development of interdigitating DC, we assessed NFrBmel protein expression and activity in an in vitro system that models DC activation and maturation. Mutie Langedams cell-like DC [immatum fetal skin-derived DC (FSDDC-I)] were expanded in primary cultures and isolated as previously described (I. Immunol. 159: 2693, 1997). In some expaiments, FSDDC-I were allowed to develop into &Is resembling mature interdigitating DC (FSDDC-M) during a several day subculture period. and in others maturation was induced by LPS (100 ng/mI). Immunofluorescence microscopy studies revealed litde if any ReIB in FSDDC-I, although RelA was detected. In contrast, RelB staining was intense in FSDDC-M and was localized to the nucleus. Quantitation of NFrSlRel proteins in cytoplasm plus nuclear FSDDC lysates (II&) by immunoblotting demonstrated a -20-fold induction of RelB during DC maturation. Similar amounts of RelB mRNA were detected in FSDDC-I and FSDDC-M by nonhero analysis, suggesting post-transcriptional regulation of RelB. Characterization of NFrB/Rel complexes by electrophoretic mobility shift assay (II+ showed that levels of active NF&/Rel hctcrodimen m FSDDC-M were 2.3-fold higher than in FSDDC-I. Almost alI NFrBiRel heterodimers in FSDDC-I contained RelA while NF&/Rel heterodimers in FSDDC-M were R&3 predominant. Finally, time course studies in which maturation was induced by LPS revealed coordinate upregulation of RelB, MHC class II and CD86. RelB may play an impatant role in DC activationlmaturation. Our in vitro model system should facilitate definitive studies of RelB metabolism and function in DC.