human dendritic cells handling of binding, uptake and degradation of free and igg-immune complexed...

Immunology 1997 90 138–146

Human dendritic cells handling of binding, uptake and degradation of free andIgG-immune complexed dinitrophenylated human serum albumin in vitro

M. LARSSON, J. BERGE, A. G. JOHANSSON & U. FORSUM Department of Clinical Microbiology, Faculty of HealthSciences, Linkoping University, Sweden

SUMMARY

The handling of free and IgG-complexed dinitrophenylated human serum albumin (DNP-HSA)by human dendritic cells (DC ) cultured with granulocyte–macrophage colony-stimulating factor(GM-CSF ) and interleukin-4 (IL-4 ) was studied. It has been shown that the amount ofuncomplexed or IgG-complexed antigen required by DC to start an immune response is lowcompared with other antigen-presenting cells. We therefore examined whether such efficientpresentation of immune complexes is due to an enhanced FccRII-mediated endocytosis or to aspecialized and efficient antigen handling, i.e. macropinocytosis. The FccRII expression was foundto be heterogeneous on the GM-CSF- and IL-4-cultured DC, i.e. it ranges from low to highexpression. The handling of antigen and immune complexes revealed, that the level of bindingand uptake of IgG–DNP-HSA complexes by in vitro expanded DC is low compared with freeantigen. Uncomplexed DNP-HSA is probably handled either by endocytosis via receptors beingmore abundant and/or efficient than the FccRII or via non-receptor-mediated endocytosis. Thebinding and uptake of IgG-complexed DNP-HSA was blocked by anti-FccRII antibody, indicatingthe specificity of the interaction.

INTRODUCTION molecules that allows presentation to more T-cell epitopes,third the high expression of adhesion and costimulatory

Human dendritic cells (DC ) are derived from CD34+ cells inmolecules, and finally low surface charge.7–9bone marrow, as well as some other myeloid cell lineages.1,2

The granulocyte–macrophage colony-stimulating factorDC are CD45+ leucocytes and highly specialized antigen-(GM-CSF) is crucial for DC proliferation from progenitorspresenting cells. They thus process and present antigens veryin different species. Cytokines, such as tumour necrosis factor-efficiently to T lymphocytes in situ, and stimulate responsesa, stem cell factor, or interleukin-4 (IL-4) are also essentialfrom naive and memory T cells in the paracortex areas ofand achieve together with GM-CSF efficient proliferation ofsecondary lymphoid organs.3,4 Dependent on the maturationthe DC progenitors. The optimal blend of cytokines for DCstage, studies in vivo and in vitro suggest, that DC exhibit someproliferation depends on the species and whether they aredifferences in phenotype, antigen capture, antigen processing,isolated from bone marrow, cord blood, or adult blood.5,10–12and immune stimulatory function.5,6Progenitors of human DC are present in bone marrow, cordSeveral mechanisms contribute to the efficient antigenblood and peripheral blood.1,2,5,11,12presentation by the DC. First, their remarkable capacity to

When IgG-complexed antigens bind to surface receptorscluster T-cells in an antigen-dependent way, second the highsuch as FccII receptors, endocytosis is induced, followed byexpression of major histocompatibility complex (MHC)efficient delivery to the MHC class II-positive processingcompartments (MIIC ), the site for antigen degradation andReceived 10 June 1996; revised 8 September 1996; acceptedpeptide loading onto MHC class II molecules.13,14 Sallusto29 September 1996.et al.13 found that cytokine-driven human DC use two distinct

Abbreviations: BSA, bovine serum albumin; DC, dendritic cells; mechanisms for antigen capture, high level of fluid phaseDNP-HSA, dinitrophenylated-human serum albumin; GM-CSF, gran-

uptake via macropinocytosis that allow continuous internaliz-ulocyte–macrophage colony stimulating factor; GPS, guinea-pigation of large volumes of fluid and through lectin–carbo-serum; HEPES, N-2-hydroxy-ethyl-piperazine-N∞-2-ethansulphonichydrate interaction via the mannose receptor.acid; IL-4, interleukin-4; LC, Langerhans cells; MHC, major histability

FccRII is expressed on many cell types, including macro-complex; MIIC, MHC class II-positive processing compartment;MLR, mixed lymphocyte reaction. phages, neutrophils, basophils, B cells, platelets, endothelial

cells and placental trophoblasts. Several isoforms exist bothCorrespondence: Dr M. Larsson, Department of Clinicalin human and mouse.15 Langerhans cells (LC) in mice, forMicrobiology, Faculty of Health Sciences, Linkoping University,

S-581 85 Linkoping, Sweden. instance, express one isoform of FccRII, and two isoforms of

© 1997 Blackwell Science Ltd138

Dendritic cell handling of free and immune-complexed antigen 139

FccRIII.16 The low-affinity receptors, FccRII and FccRIII cultured in RPMI medium containing 5% human serum in96-well microtitre plates with 250, 500, 1000, 2000, 4000, orexpressed by mouse LC, may have biological relevance, since

FccR mediated-endocytosis increases the efficiency of antigen 8000 dendritic cells. [3H]Thymidine incorporation was meas-ured on day 4 or 5 after a 16 hr pulse with [3H]thymidinepresentation to T cells.16 The expression of FccRII, FccRIII

( trace) and C3R (CD11b/CD18) on freshly isolated DC from (1 mCi/well, Amersham Life Science, Buckinghamshire, UK).human peripheral blood, LC, and cytokine-driven DC hasbeen demonstrated in several studies.5,17,18 Antigens incorpor- Preparation of DNP-HSA and anti-DNP antibodiesated in immune complexes presented to in vitro expanded Lyophilized human serum albumin (HSA, fraction V;human DC and mouse LC elicit immune responses at much Calbiochem Co. La Jolla, CA) was dissolved in phosphate-lower antigen concentrations, compared to antigen alone.5,16 buffered saline (PBS), pH 7·6, and conjugated with an average

The importance of IgG on binding, uptake and degradation of 10 dinitrophenyl (DNP) groups per HSA molecule asof antigen by cytokine-driven human DC has not been investi- described previously.19 The DNP-HSA preparation was lab-gated. The aim of this study was therefore to characterize and elled with 125I coupled to tyramine cellobiose20 or labelledquantify cell association of dinitrophenyl-human serum albu- with FITC. Rabbit anti-DNP immune serum was prepared bymin (DNP-HSA) and IgG–DNP-HSA complexes at 4° or at Dakopatts (A/S Glostrup, Denmark), rabbits were immunized37° by human DC, respectively, and also to quantify the with DNP-keyhole limpet haemocyanin (Calbiochem Co.).degradation of IgG–DNP-HSA complexes and DNP-HSA in IgG anti-DNP antibodies were isolated by affinity chromatog-these cells. The results were compared with results obtained raphy21 and gel permeation chromatography (FPLC, Superoseusing tissue macrophages (rat Kuppfer cells). The binding and 6, Pharmacia). The purity was controlled by double radialuptake of IgG–DNP-HSA complexes by DC were found to immunodiffusion. The IgG anti-DNP preparation was concen-be FccRII-mediated at a low but specific level. The amount trated by hyperosmotic dialysis and then extensively dialysedtaken up should be sufficient to elicit specific T-cell against PBS pH 7·6. The IgG concentration was determinedstimulation.5,16 spectrophotometrically at 280 nm.

MATERIALS AND METHODS Uptake and binding of DNP-HSA or IgG/DNP-HSA bydendritic cellsCulture mediumThe IgG immune complexes were formed at 10-fold molarWe used RPMI-1640 (Gibco Laboratories, Grand Island, NY )antibody excess. The 125I-labelled DNP-HSA (44 n) wassupplemented with 200 m -glutamine, 20 mg/ml gentamycin,dissolved in 0·2 N-2-hydroxy-ethyl-piperazine-N∞-2-ethan-10 m HEPES, and 10% heat-inactivated fetal calf serumsulphonic acid (HEPES) buffer, and incubated with IgG anti-(FCS ), as well as the recombinant human cytokines, GM-DNP antibodies for 45 min at 37°. The IgG–DNP-HSACSF (Schering-Plough/Sandoz, Keniworth, NJ ) and IL-4immune complexes were characterized by ultracentrifugation(Genzyme Corp., Cambridge, MA).in a continuous sucrose gradient (5–45%). The samples werecollected by upward displacement using 65% sucrose solution.Culture of dendritic cells from blood of healthy donorsThe DNP-HSA solution was prepared in HEPES buffer withThe method described by Romani et al. was used with modi-2% bovine serum albumin (BSA) (fraction V, Boehringerfication.12 Mononuclear cells were isolated from buffy coatsMannheim, Germany), the IgG–DNP-HSA solutions wereby flotation on Ficoll–Hypaque (1·0777 g/ml, Pharmacia,prepared in HEPES buffer, normal guinea-pig serum (GPS )Uppsala, Sweden), depleted of T cells by rosetting with neura-or heat-inactivated GPS. DC were preincubated in 0·2 minidase-treated sheep red blood cells and then culturedHEPES buffer containing 2% BSA for 30 min at 37°. Theat 0·5–0·7 106 cells/ml in the culture medium containingIgG–DNP-HSA immune complex or DNP-HSA solutions1000 U/ml GM-CSF and 700 U/ml IL-4. After 8 days ofwere pipetted into the test tubes giving a final concentrationculture the DC were examined by mixed lymphocyte reactionof 106 cells in 1% BSA and 44 n of DNP-HSA in each tube.(MLR) and fluorocytometry.Incubation was futher done under gentle agitation at 37°.After 10, 30, 60, 120, or 300 min, tubes of either kind werePhenotypic characterization by cytofluorographic analysis:centrifuged for 3 min and then washed three times in ice-coldfluorocytometryHEPES containing 1% BSA. The radioactivity was measuredPhenotypic analysis of the dendritic cells was performed usingin each sample in a gamma-counter (Compugamma 1282an EPICS Profile I flow cytometer with Power Pac (CoulterLKB, Wallac). The binding experiments were carried out atElectronics Corporation, Hialeha, FL). DC were stained with4°, to avoid internalization, but in all other aspects as describedspecific mouse monoclonal antibodies (Fig. 1) [kind gift of Drabove for the uptake experiments. Binding and uptake experi-N. Bhardwaj, Rockefeller University, New York or obtainedments were performed three times using DC from differentfrom Dakopatts, Copenhagen, Denmark; Serotec (Oxford,cell cultures.UK ) or Becton Dickinson (Mountain View, CA)] followed

by fluorescein isothiocyanate (FITC)-conjugated mouse anti-mouse F(ab)2 fragments (Dakopatts) diluted 15100. Preparation of rat Kupffer cells

Rat Kupffer cells were prepared as described by Johansson &Skogh.22 Briefly, isolated rat liver cells were obtained byFunctional analysis by MLR

Allogenic primary mixed lymphocyte reactions were performed collagenase perfusion of the liver. Kupffer cells were isolatedfrom the liver cell suspension by differential centrifugationusing T cells purified from leucocyte-enriched buffy coats and

cultured dendritic cells. The T cells (2×105 cells/well ) were and elutriation.

© 1997 Blackwell Science Ltd, Immunology, 90, 138–146

M. Larsson et al.140

Figure 1. Histograms from one representative cytoflourographic analysis of dendritic cells, isolated from human peripheral bloodand expanded with GM-CSF and IL-4. Phenotypic analysis of the dendritic cells was done by staining with different mousemonoclonal antibodies followed by FITC-conjugated rabbit anti-mouse F(ab)2 fragments. The mAb cocktail contained anti-CD3,anti-CD14, anti-CD19 and anti-CD56.



Binding and uptake of DNP-HSA or IgG–DNP-HSA by rat RESULTSKupffer cells

Characterization of in vitro driven dendritic cells from humanThe binding and uptake experiments with rat Kupffer cells

peripheral bloodwere performed as described above for DC, but the DNP-HSA concentration was 22 n in these experiments. Binding Cultured progenitor cells, i.e. mononuclear cells obtained after

Ficoll–Hypaque separation of buffy coats and depleted ofand uptake were performed three times using Kupffer cellsfrom different rats. T cells, first created aggregates comprising two or three cells,

but were futher expanded to larger then 10 cells in diameter.Typical veiled cells were visible at the rim of the aggregatesDegradation of DNP-HSA or IgG–DNP-HSA by dendritic cells

The degradation of DNP-HSA or IgG–DNP-HSA in vitro by and on single cells released from the aggregates. After 8 daysof culture, the cytokine-driven DC were harvested and theDC was estimated by mixing equal volumes of the DC

suspension with 10% trichloroacetic acid (TCA). The mixture stimulatory capacity and the phenotype determined.Examination of the DC phenotype by flourocytometrywas incubated for 2 hr at 4°. After centrifugation the radio-

activity was measured in the pellet (=non-degraded ligand) (Fig. 1) revealed that the cytokine-expanded DC had charac-teristic expression of MHC class II molecules, CD1a, CD4,and in the supernatant (=degraded ligand). Degradation was

expressed as per cent of total cellular uptake of DNP-HSA or CD11a, CD11b, CD11c, CD15s, CD18, CD29, CD32, CD44,CD54 and CD58, but did not display CD3 (T cell ), CD14IgG–DNP-HSA immune complexes by the different DC

fractions. (monocyte/macrophage) and CD19 (B cell ). CD14 expressionwas negative or dim in the different experiments. FccRII(CD32) immunofluorescence staining of the DC revealed simi-Immunofluorescence staining of FccRII on dendritic cells

Microscope specimens were prepared in a cytocentrifuge lar heterogeneity as the cytofluorographic analysis (Fig. 1).The DC differ in their expression of FccRII from almost(Shandon Southern Instruments Inc., Sewickley, PA) using

20×104 to 30×104 cells/slide. After fixation for 5 min in negative to very high expression (Fig. 2).The functional capacity of the cytokine-expanded DC wasacetone the DC were washed three times in PBS with 1% BSA

and incubated for 30 min at room temperature with mouse examined by the ability to stimulate T cells in allogen mixedlymphocyte reactions. Fixed number of T cells were coculturedmonoclonal antibody against FccRII proteins (AT10, Serotec),

diluted 1550 in PBS with 1% BSA. The DC were washed three in 5% HS RPMI medium with a range from 250 to 8000 DC.Figure 3 shows data from one representative experiment.times for 3 min in PBS with 1% BSA. FITC-conjugated rabbit

anti-mouse F(ab)2 fragments (Dakopatts) diluted 15100 inPBS with 1% BSA were added and the cells were further

Characterization of IgG mmune complexesincubated for 30 min at room temperature. The slides wererinsed as described above. The CD32-stained DC were incu- The IgG immune complexes formed in HEPES buffer were ofbated with 6×10−7 propidium iodine for 15 seconds. varying sizes and about 80% contained two or more antibodies.

The immune complex preparation comprised only minorEndocytosis of antigen amounts of uncomplexed DNP-HSA (Fig. 4). The IgG–DNP-DC were incubated for 10, 30, or 60 min at 37°, with HSA preparations which were formed in normal or in heat-0·66 mg/ml DNP-FITC-HSA (DNP/HSA and FITC/HSA inactivated GPS were larger then the ones formed in HEPES.molecular ratio were both 9·8). Endocytosis was stopped by IgG immune complexes formed in normal GPS were slightlyadding excess of ice-cold RPMI. The cells were washed three larger compared with the IgG immune complexes formed intimes by centrifugation at 4°, and then chased at 37° for 0, heat-inactivated GPS (Fig. 4).10, 20, 40, or 60 min to allow further endocytosis andexocytosis. Cytospins were prepared at all different times. TheDNP-FITC-HSA endocytosis was examined with confocalmicroscopy, using a Phoibos 2000 (Molecular Dynamics,Sunnyvale, CA).

FccII receptor blocking of the binding and uptake ofIgG–DNP-HSA immune complexes with anti-CD32 antibodyDC were preincubated with excess (30 mg/ml ) of FccR IIblocking anti-CD 32 antibody (AT10, Serotec) for 1 hr at 37°in HEPES with 2% BSA and then washed twice in ice-coldRPMI. In the control was the CD32 antibody omitted butotherwise performed as above. The preincubated DC wereincubated with IgG–DNP-HSA immune complexes preparedin HEPES at 37° for 30, 60 and 120 min, and then washedthree times in ice-cold RPMI, and cytospin samples were

Figure 2. Fluorescence micrograph showing two cytokine-driven den-prepared. The IgG–DNP-HSA immune complexes were dritic cells with high expression of CD32. The cells were stained withstained with FITC-conjugated anti-HSA rabbit IgG mouse monoclonal antibody raised against CD32 proteins followed(Dakopatts) diluted 1525 for 30 min at room temperature and by FITC-conjugated rabbit anti-mouse F(ab)2 fragments and thenwashed for 3 min thrice with PBS–BSA. The slides were incubated in propidiumiodine to visualize all cells including negative

contaminating cells and weakly stained cells. Magnification ×800.examined with fluorescence microscopy.



10 min at 37° was five to six times higher than the binding at4° and increased with time.

The DC uptake of IgG–DNP-HSA immune complexesprepared in normal GPS was about the same as for IgG–DNP-HSA prepared in heat-inactivated GPS (Fig. 5d), but slightlylower for IgG–DNP-HSA prepared in HEPES (Figs. 5c,d).However, the amount of uncomplexed DNP-HSA endocytosedby the DC was two to three times higher, compared to theDNP-HSA complexed with IgG in either way (Figs. 5c,d ).

Binding and uptake of DNP-HSA and IgG–DNP-HSA by ratKupffer cells

Figure 3. T-cell stimulatory function (primary allogenic MLR) ofhuman DC grown from peripheral blood with GM-CSF and IL-4. In order to evaluate the relative capacity of the in vitroResponder cells were T lymphocytes (2×105/well ). The MLR was expanded DC ability to bind and ingest DNP-HSA and IgG–pulsed with [3H]-thymidine (1 mCi/well ) for 16 hr at day 5. DNP-HSA, they were compared to rat Kupffer cells in a well-

characterized system.23 In contrast to DC, is IgG–DNP-HSAimmune complex binding by rat Kupffer cells higher comparedto uncomplexed DNP-HSA binding (Fig. 6a). Thus, Kupffercell-binding of DNP-HSA at 4° is two to three times higherthan the DC binding of DNP-HSA (Figs. 5a,6a). Interestingly,the binding of immune complexes by Kupffer cells was about70- to 80-fold higher than by the DC. IgG thus clearlyenhanced the adhesion and uptake of DNP-HSA to rat Kupffercells (Fig. 6b). The amount of uncomplexed DNP-HSA anti-gen taken up by Kupffer cells was only three to four timeshigher than for DC and the uptake of IgG–DNP-HSA pre-pared in HEPES was 15 times as efficient as the uptake for DC.

Endocytosis of FITC-DNP-HSA

Endocytosis of FITC-DNP-HSA by DC was visible alreadyafter 2–4 min of incubation (data not shown). Figure 7 showsa confocal image of endocytosed DNP-HSA inside DC whenincubated for 30 min with and then chased for 40 min withoutexogenous free antigen.

Figure 4. Characterization of IgG–DNP-HSA immune complexes byultracentrifugation in a continuous sucrose gradient (5–45%). The top

Degradation of DNP-HSA or IgG–DNP-HSA by dendritic cellsof the gradient is to the left and fraction 25 is the cut bottom of thetube.The immune complex preparation contained only negligible Degradation of DNP-HSA and IgG–DNP-HSA at 60 min byamounts of uncomplexed DNP-HSA. Fraction nr, fraction number. the DC, as evidenced by material not precipitated with TCA

precipitation, was around 5% of the total cell-associated DNP-HSA or IgG–DNP-HSA prepared either in HEPES, normal

Binding of DNP-HSA or IgG–DNP-HSA immune complexesGPS or in heat-inactivated GPS (Table 1).

to dendritic cells at 4°Figure 5(a) shows the binding of DNP-HSA and IgG–DNP-

Anti-CD32 antibody blocks the uptake of IgG–DNP-HSA byHSA prepared in HEPES buffer to DC. There was no big

dendritic cellsdifference between DNP-HSA found on the DC compared toIgG–DNP-HSA. Formation of IgG–DNP-HSA in normal The DC were preincubated with an excess of anti-CD32

antibody to block FccRII-mediated immune complex inges-GPS did not increase the binding to the DC, compared toIgG–DNP-HSA formed in heat-inactivated GPS (Fig. 5b). tion. The CD32 antibody is anticipated to recognize the Fc

part of FccRII and block further binding. Thus DC that wereMoreover, the cell association of IgG–DNP-HSA formed innormal GPS and of IgG–DNP-HSA formed in HEPES buffer preincubated with anti-CD32 antibody and subsequently incu-

bated with IgG-immune complexes, showed no, or very low,by the DC were nearly the same (7 ng/106 DC; Figs. 5a,b).binding and uptake of IgG immune complexes (Fig. 8b) ascompared with DC not preincubated with anti-CD32 antibody

Uptake of DNP-HSA or IgG–DNP-HSA by dendritic cells(Fig. 8a). DC incubated only with immune complexes showed

at 37°the same pattern of binding as DC stained for CD32 (Fig. 3),i.e. a heterogeneous distribution of IgG–DNP-HSA immuneThe uptake of DNP-HSA by DC at 37° was two to three

times higher than that of IgG–DNP-HSA prepared in HEPES complexes. The different incubation times 30, 60 and 120 minrevealed only slight differences in IgG–DNP-HSA binding and(Fig. 5c). Futhermore, the amount DNP-HSA ingested after



Figure 5. Binding and uptake of DNP-HSA or IgG–DNP-HSA immune complexes by cytokine-driven DC. The data are obtainedfrom three different experiments using different cultures of cytokine-driven DC. (a) The DNP-HSA (%) and IgG–DNP-HSAimmune complexes (&) prepared in HEPES binding to DC at 4°. (b) The binding by DC at 4° of IgG–DNP-HSA immunecomplexes prepared in normal GPS (%) and IgG–DNP-HSA immune complexes prepared in heat-inactivated GPS (&). (c) Theuptake of DNP-HSA (G ) and IgG–DNP-HSA prepared in HEPES (&) by DC at 37°. (d ) The uptake by DC at 37° of IgG–DNP-HSA immune complexes prepared in normal GPS (%) and IgG–DNP-HSA prepared in heat-inactivated GPS (&).

uptake by the DC. These results are similar to the findings these cells. We futhermore investigated whether the bindingshown in Figs 5(c) and 5(d) for immune complexes. and uptake of IgG–DNP-HSA was mediated by FccRII. The

characterization of the cellular handling of DNP-HSA andIgG–DNP-HSA is of interest, since recent findings haveDISCUSSIONrevealed that antigen administered in the form of IgG immune

In this report DC was expanded in vitro and we quantified the complexes, to mouse LC and cytokine-driven human DC, arebinding and uptake of DNP-HSA and IgG–DNP-HSA to more efficient to stimulate a T-cell response, compared with

uncomplexed antigen.5,16 In vivo and in vitro studies haveTable 1. Degradation of DNP-HSA and IgG–DNP-HSA immune suggest that, dependent on the maturation stage, DC exhibitcomplexes by dendritic cells at 60 min of uptake. The average of three some differences in phenotype, antigen capture, antigen-

experiments is shown processing, and immune-stimulating capacity.5,6 Furthermorethe MHC class II-negative DC progenitor in blood is moreDegradation, % of total uptakedifferentiated than the CD34+ progenitor in bone marrow.24after 60 minIt is, however, now possible to achieve proliferation anddifferentiation of dendritic cells progenitors in vitro, by cultur-DNP-HSA 5·3

IgG–DNP-HSA prepared in 5·3 ing the cells in the presence of GM-CSF and IL-4. This resultsHEPES in DC with phenotype and functions similar to in vivo imma-

IgG–DNP-HSA prepared in 4·4 ture DC.5,12normal GPS Most of myeloid progenitors in blood develop into macro-

IgG–DNP-HSA prepared in 5·6phages rather than DC unless their potential for monocyteheat-inactivated GPSdifferentiation is suppressed by IL-4.5,25 Earlier methods used



Figure 8. Fluorescence micrographs showing blocking of IgG–DNP-HSA immune complex binding and uptake by DC at 37° by anti-FcRII (CD32) antibody. The dendritic cells were preincubated with access

Figure 6. Binding and uptake of DNP-HSA and IgG–DNP-HSA by of FcR blocking anti-CD 32 antibody and then followed by incubationrat Kupffer cells. The data are obtained from three different experi- with IgG–DNP-HSA immune complexes at 37° for 120 min andments using Kupffer cells from different rats. (a) The IgG–DNP-HSA visualized by immunofluorescence staining with FITC-conjugated anti-immune complex (&) and DNP-HSA (%) binding by rat Kupffer HSA rabbit IgG antibody. (a) The control DC, not preincubated withcells at 4°. (b ) The IgG–DNP-HSA immune complex (&) and DNP- anti-CD32 antibody showed binding and uptake of IgG–DNP-HSAHSA (%) uptake by rat Kupffer cells at 37°. immune complexes, (b) while DC preincubated with anti-CD32 anti-

body showed no or much less binding and uptake of IgG–DNP-HSAimmune complexes.

to isolate human DC have resulted in populations with thephenotype and functions of mature DC. The DC generatedfrom our in vitro cultures had the same phenotype and T-cellstimulatory capacity, as the DC described by Sallusto &Lanzavecchia5 and Romani et al.12 The identification of thesecells as DC is based on typical morphology and motility,appropriate surface phenotype and remarked high efficiencyto stimulate naive T cells.26–28

Our cytokine-driven DC expressed high levels of comple-ment receptors, i.e. CR3 (CD11b/CD18) and CR4 (CD11c/CD18). However, in our study we found no evidence for anyparticipation of complement in the uptake of IgG immunecomplexes by DC, as judged by the experiments performedusing IgG immune complexes prepared in normal or heat-inactivated GPS.

The DC population expanded with cytokines expressFigure 7. Confocal image showing endocytosis of FITC-DNP-HSAFccRII.5 Both our immunofluorescence staining and cyto-by cytokine-driven human DC. DC were incubated for 30 min at 37°fluorographic analysis revealed heterogeneity of FccRIIwith FITC-DNP-HSA and then chased for 40 min to allow further

endocytosis. expression ranging from low to very high (Figs. 1 and 2). The



heterogeneity among the DC could depend upon when they for immune complex binding and uptake. As a consequence,variation in FccRII expression between our different prep-developed in the cytokine culture. FccRII is the most widely

distributed FccR, but it has low affinity for IgG and can only arations of DC may explain the differences in binding anduptake of immune complexes.interact well with IgG immune complexes or IgG-coated

particles. The affinity of FccRII can, however, be up-regulated Macropinocytosis is constitutive in DC days after removalof GM-CSF and IL-4, and can be detected in fresh veiledby proteolysis.15 Receptor-mediated endocytosis involves local-

ization of immune complexes to clatrin-coated pits, whereas cells.13 A calcium-dependent mannose receptor has been ident-ified as the major receptor for FITC-dextran and horseradishinternalization by phagocytosis is dependent upon intact actin

microfilaments.29 The formation of immune complexes is a peroxidase (HRP) endocytosis by human dendritic cells.13Since the mannose receptor is known to bind also hydrophobicphysiological means to eliminate antigen and to regulate

immune responses.30 Cross-linking of FccRII on monocytes molecules, DNP-HSA which is hydrophobic could be handledthe same way as the FITC-dextran and the HRP.35and granulocytes by immune complexes results in effector

responses such as phagocytosis, antibody-dependent cellular Our results indicate that the binding and uptake of immunecomplexes in DC is a specific event, but with low efficiency,cytotoxicity and release of inflammatory mediators.30

The human FccRII isoforms FccRIIb1 and FccRIIb2 on due to a limiting factor, most probably the amount of FccRIIreceptors. Since binding and uptake of IgG-complexed DNP-transfected COS-1 cells, do not induce phagocytosis, but are

able to bind soluble immune complexes and mediate endo- HSA was blocked by anti-FccRII antibody, this futher streng-thens that the handling of IgG immune complex is FccRII-cytosis.29 Mouse LC are able to internalize IgG-containing

immune complexes via their FccRII.16 In mice, the FccRIIb2 mediated. The level of IgG immune complex uptake is prob-ably sufficient to give a high and specific T-cell-mediatedisoform can internalize through receptor-mediated endo-

cytosis, whereas FccRIIb1 isoform can only cap the recep- immune response. In DC it is not the amount of antigen/immune complex handled, instead it is the efficiency in pro-tor.31,32 The isoforms of FccRII expressed by the human DC

are not known. One may speculate that they could be the cessing and presenting antigens, that makes the DC an efficientantigen-presenting cell.same type as described for the mouse LC, i.e. isoforms with

the capacity to internalize through receptor-mediatedendocytosis, corresponding to FccRIIb1 and FccRIIb2 in ACKNOWLEDGMENTShumans.16,29

This study was supported by grants from the Swedish society forThe immune complexes used in this study, ranged mostlymedical research, The Ake Wiberg foundation and The Welanderfrom dimeric to large immune complexes (Fig. 3) consideredfoundation.to represent authentic and well-characterized immune com-

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