Capacity of Human Dendritic Cells Uptake Receptor Expression And

The Novel Cyclophilin-Binding Drug Sanglifehrin A Specifically Affects Antigen Uptake Receptor Expression and Endocytic Capacity of Human Dendritic Cells This information is current as of September 25, 2021. Andrea M. Woltman, Nicole Schlagwein, Sandra W. van der Kooij and Cees van Kooten J Immunol 2004; 172:6482-6489; ; doi: 10.4049/jimmunol.172.10.6482 http://www.jimmunol.org/content/172/10/6482 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Novel Cyclophilin-Binding Drug Sanglifehrin A Speciﬁcally Affects Antigen Uptake Receptor Expression and Endocytic Capacity of Human Dendritic Cells1

Andrea M. Woltman,2 Nicole Schlagwein, Sandra W. van der Kooij, and Cees van Kooten

Sanglifehrin A (SFA) is a recently developed immunosuppressant that belongs to the family of immunophilin-binding ligands. SFA is a cyclophilin A-binding immunosuppressive drug with a novel, but unidentiﬁed, mechanism of action. Several reports exist about the effect of SFA on T cells, but its effect on the initiators of the immune response, i.e., dendritic cells (DCs), is relatively unknown. Therefore, we examined the effect of SFA on the differentiation and function of human monocyte-derived DCs. Unlike the well-known cyclophilin A-binding immunosuppressant cyclosporin A, which did not affect DC phenotype, differentiation of

DCs in the presence of SFA resulted in CD14-CD1a DCs with normal DC morphology, viability, and a proper capacity to activate Downloaded from allogeneic T cells. However, DCs generated in the presence of SFA demonstrated reduced macropinocytosis and lectin-mediated endocytosis, which was in line with a decreased expression of C-type lectins, including mannose receptor, C1qRP, DC-ASGPR, and especially, DC-SIGN. In contrast, Fc␣RI (CD89) and Fc␥RII (CD32) were increased by SFA. The explicit effect of SFA on the expression of Ag uptake receptors and Ag capture by DCs makes SFA unique among immunophilin-binding immunosuppressive drugs. The Journal of Immunology, 2004, 172: 6482Ð6489. http://www.jimmunol.org/ endritic cells (DCs)3 are the most potent APCs. They are and immunogenic responses, both processes provide a target for highly motile cells that operate at the interface of innate the action of immunosuppressive drugs. Immunophilin-binding D and acquired immunity with a key role in the initiation immunosuppressive drugs, including cyclosporin A (CsA), and direction of immune responses (1, 2). DCs constitutively pa- FK506, and rapamycin (RAPA), initially selected for their effects trol through the blood, peripheral tissues, lymph, and secondary on T cells, also have profound immunomodulatory effects on DC lymphoid organs. In peripheral tissues, DCs act as sentinels against function. RAPA induces strong apoptosis in DCs (6Ð8) and im- foreign Ags, but they can also take up tissue-derived self Ags, pairs Ag uptake via both receptor-mediated endocytosis and mac- which plays an important role in tolerance induction and mainte- ropinocytosis, whereas the calcineurin-inhibitors CsA and FK506 by guest on September 25, 2021 nance to self Ags (3). demonstrated some effects on DC maturation without affecting Efficient Ag internalization, either through receptor-mediated their survival or Ag uptake capacity (8Ð11). endocytosis or fluid-phase endocytosis (i.e., macropinocytosis) is a Sanglifehrin A (SFA) is a recently developed immunosuppres- specific attribute of immature DCs. Macropinocytosis represents a sive macrocyclic compound produced by the actinomycetes strain critical Ag uptake pathway allowing DCs to rapidly and nonspe- Streptomyces sp. A92-308110 (12, 13). Like CsA, SFA binds to cifically sample large amounts of surrounding fluid (4). Receptor- the immunophilin cyclophilin A but with an affinity that is 20-fold mediated endocytosis, in contrast, is initiated by the engagement of higher than that of CsA (12). Like other immunophilin-binding specific receptors, triggering a cascade of signal transduction that drugs, the immunosuppressive activities of SFA and its mechanism is required for actin polymerization and effective engulfment. Re- of action have been mostly studied on T cells. The cyclophilin- ceptors for Ag capture, such as C-type lectins and Fc receptors, SFA complex does not interact with calcineurin and does not affect vary in their ligand specificity and mode of delivery to Ag-pro- calcium-dependent IL-2 production (14). Rather, it has been dem- cessing compartments. In addition, depending on the route of in- onstrated to inhibit IL-2-dependent T cell proliferation, which re- ternalization and the source of Ag, the internalization process can sembles the action of RAPA (15). Furthermore, SFA shows some lead to DC maturation and subsequent T cell activation (5). inhibitory activities on IgG production by B cells, on TNF-␣ pro- Because Ag uptake and presentation together with the regulation of costimulation are essential for the induction of both tolerogenic duction by monocytes (14), and on IL-12 production by DCs (16). In the present study the effect of SFA on human DC differentiation and function was investigated in detail. SFA influenced the Department of Nephrology, Leiden University Medical Center, Leiden, The Nether- differentiation of human monocyte-derived DCs resulting in cells lands that normally lost their expression of CD14 and lacked the ex- Received for publication October 3, 2003. Accepted for publication March 10, 2004. pression of CD1a. Importantly, DCs generated in the presence The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance of SFA demonstrated reduced macropinocytosis and lectin-me- with 18 U.S.C. Section 1734 solely to indicate this fact. diated endocytosis, which was in line with a decreased expres- 1 A.M.W. is supported by Grant C01.1970 from the Dutch Kidney Foundation. sion of C-type lectins, such as mannose receptor (MR) and DC- 2 Address correspondence and reprint requests to Dr. Andrea M. Woltman, Depart- specific ICAM-3-grabbing nonintegrin (SIGN). In contrast, ment of Nephrology, C3-P, Leiden University Medical Center, Albinusdreef 2, 2333 Fc␣RI (CD89) and Fc␥RII (CD32) were increased by SFA. Its ZA Leiden, The Netherlands. E-mail address: [email protected] explicit effect on the expression of Ag uptake receptors and Ag 3 Abbreviations used in this paper: DC, dendritic cell; SIGN, specific ICAM-3-grabbing nonintegrin; SFA, Sanglifehrin A; CsA, cyclosporin A; LY, Lucifer yellow; MR, capture makes SFA unique among immunophilin-binding im- mannose receptor; RAPA, rapamycin. munosuppressive drugs.

Materials and Methods independent experiments. Mean fluorescence intensities were analyzed Reagents with Student’s t test for paired samples. Results were considered significant if p Ͻ 0.05. SFA was provided by Novartis Pharmaceuticals (Basel, Switzerland). A stock solution (10Ϫ3 M) of SFA was made in methanol, which was further Results diluted in medium directly before use at the concentrations indicated. CsA Effects of the cyclophilin-binding compounds CsA and SFA on (Neoral, Novartis Pharmaceuticals) and RAPA (Calbiochem, Cambridge, MA) were used at 10Ϫ6 M final concentration. Propidium iodide was pur- DC morphology and phenotype chased from Molecular Probes (Leiden, The Netherlands) and used at 10 Monocytes were cultured in the presence of IL-4 and GM-CSF. ␮g/ml final concentration. After 6 days of culture, a nonadherent cell population with pro- Generation monocyte-derived DCs truding veils typical for DCs was obtained. When similar cultures DCs were generated as previously described (9). In brief, human mono- were performed in the presence of the cyclophilin-binding drugs Ϫ6 Ϫ6 cytes were isolated from buffy coats obtained from healthy donors using CsA (10 M) or SFA (10 M), or the solvent methanol, cells Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO) and Percoll (Pharmacia showed a comparable DC morphology (Fig. 1A). Addition of SFA Biotech, Uppsala, Sweden) density gradient centrifugation, followed by or methanol did not affect cell yields, whereas CsA slightly decreased plastic adherence (2 h). DCs were generated in 6-well culture plates cell survival as determined by trypan blue exclusion (Fig. 1B). (Costar, Cambridge, MA) in RPMI 1640 containing 10% heat-inactivated FCS and penicillin/streptomycin supplemented with 5 ng/ml GM-CSF DCs, either cultured under normal conditions or with CsA, dem- (Leucomax, Novartis Pharmaceuticals) and 10 ng/ml IL-4 (PeproTech, onstrated the typical DC phenotype: both CD1a and DC-SIGN Rocky Hill, NJ) for at least 6 days. SFA-DCs were generated by addition were clearly induced, CD14 was completely down-regulated, and of SFA from day 0 (monocyte stage) onward, unless otherwise indicated. DCs demonstrated a relative low expression of CD86 and HLA- Downloaded from Analysis of cell surface molecules by FACS DR, which is characteristic for immature DCs (Fig. 2). In contrast, SFA inhibited the induction of CD1a ( p ϭ 0.005), reduced the Cells were harvested and washed in buffer containing 1% BSA, 1% heat- induction of DC-SIGN ( p ϭ 0.025), but did allow the down-reg- inactivated normal human serum, and 0.02% NaN3. FACS analysis was performed using mAbs against CD1a (Leu-6) and CD14 (Leu-M3)(both ulation of CD14. Although SFA seemed to increase the expression BD Biosciences, San Jose, CA), HLA-DR (clone B8.11,2), CD86 (IT2.2; of CD86 and HLA-DR, a significant difference compared with BD PharMingen, San Diego, CA), Fc␥RIII/CD16 (3G8), Fc␥RII/CD32 Ͼ control cultures could not be observed ( p 0.05). Thus, although http://www.jimmunol.org/ (IV.3 and AT10), and Fc␥RI/CD64 (nr.22) (all provided by J. G. J. van de Winkel, Universitair Medisch Centrum, Utrecht, The Netherlands), MR/ both CsA and SFA are known to exert their immunomodulatory CD206 (D547.3; kindly provided by F. Koning, Leiden University Medical effect via binding to cyclophilin A, only SFA does significantly Center, Leiden, The Netherlands), Fc␣RI/CD89 (2D11; see Ref. 17), DC- change the DC phenotype. SIGN/CD209 (AZN-D1, a gift of Y. van Kooyk, VU Medisch Centrum, Amsterdam, The Netherlands), DC-asialoglycoprotein receptor (ASGPR) SFA does not affect T cell stimulatory capacity of DCs in (AG-5; a gift of S. Saeland, Schering-Plough, Dardilly, France), C1qRP/ allogeneic MLR CD93 (R139; see Ref. 18 and kindly provided by A. J. Tenner, University of California, Irvine, CA). Staining was visualized by PE-conjugated goat Next, the effect of SFA on the T cell stimulatory capacity of DCs anti-mouse Ig (Dako, Glostrup, Denmark) and assessed for fluorescence was investigated in allogeneic MLRs. Addition of SFA to control intensity by flow cytometry (FACSCalibur, CellQuest software; BD Bio- by guest on September 25, 2021 sciences). Dead cells, characterized by propidium iodide uptake, were excluded from analysis. Allogeneic MLR Responder T cells used for allogeneic MLR assays were isolated by sheep erythrocyte rosetting of mononuclear cells obtained from healthy donors. Stimulator cells were irradiated (50 Gy) and added in graded doses to 1.5 ϫ 105 allogeneic T cells in 96-well U-bottom tissue culture plates in RPMI 1640 containing 10% heat-inactivated FCS (0.2 ml/well). Cell proliferation was quantified by incubating the cells during the last8hofthe5-day cultures with 1 ␮Ci (37 kBq) of [methyl-3H]thymidine (NEN, Boston, MA). Results are presented as the mean cpm Ϯ SD obtained from triplicate cultures, or as mean cpm Ϯ SEM from five independent experiments. Ag uptake assays Fluid phase endocytosis (macropinocytosis) was measured as the cellular uptake of 100 ␮g/ml Lucifer yellow (LY) dipotassium salt (Molecular Probes) or 0.2 ␮g/ml BSA-FITC (BSAFITC, 1:12 molar ratio; Sigma-Al- drich), whereas lectin-mediated endocytosis was measured as the cellular FITC uptake of 0.2 ␮g/ml ␣-D-mannosylated-BSA-FITC (manBSA , 1:2.5 molar ratio; Sigma-Aldrich) or 100 ␮g/ml dextran-FITC (dextranFITC, m.w. 40,000; Molecular Probes). Approximately 5 ϫ 104 DCs were incubated at 37¡C in the presence of culture medium containing LY or dex- tranFITC. Lectin-mediated uptake was blocked by 15 min preincubation with 50 mM D-mannose (Sigma-Aldrich). Negative controls were incubated with the respective Ag at 4¡C. Ag uptake was stopped by extensive cold washing in PBS-1% and FCS-0.02% NaN3, and cell surface fluorescence was quenched with trypan blue (Sigma-Aldrich). Ag uptake was evaluated by flow cytometry. FIGURE 1. Influence of SFA and CsA on DC morphology and cell yields. Monocytes were cultured for 6 days with IL-4 and GM-CSF under Statistical analysis control conditions (Ϫ), or in the presence of CsA (10Ϫ6 M), SFA (10Ϫ6 M), or methanol (1/10000 dilution). A, The morphology of the cells (original Phenotypic analyses of DCs cultured either with or without SFA and pre- ϫ sented as histograms are representative for at least five independent exper- magnification 200) and the relative cell yields obtained by trypan blue iments with different donors. Statistical differences in endocytic capacity of exclusion are shown. B, The mean Ϯ SEM cell recovery compared with DCs cultured either in the presence or absence of SFA (10Ϫ6 M) were control cultures of three independent experiments with different donors is analyzed after 30 min of incubation and are representative for at least six shown. 6484 SFA SPECIFICALLY AFFECTS ENDOCYTOSIS BY HUMAN DC

FIGURE 2. Inﬂuence of SFA and CsA on DC phenotype. Monocytes were cultured for 6 days with IL-4 and GM-CSF in the presence of CsA (10Ϫ6 M) or SFA (10Ϫ6 M). Cell surface expression of the indicated proteins was analyzed by ﬂow cytometry as described in Materials and Methods. Data shown are representative of six independent experiments. Downloaded from

MLRs showed a dose-dependent inhibition of T cell proliferation lectins that can be found on the surface of DCs. The decreased Ϫ8 Ϫ7 Ϫ6 FITC (IC50,10 Ð10 M)(Fig. 3A). Addition of 10 M SFA to day 6 ability of SFA-DCs to take up glycoproteins like dextran and immature DCs did not decrease their T cell stimulatory capacity manBSAFITC may be explained by the down-regulation of C-type (data not shown), which was in line with the lack of signiﬁcant lectins, as observed for the decreased expression of DC-SIGN

changes in the expression of costimulatory molecules or with LPS- (Fig. 2). Therefore, the effect of SFA on the expression of the http://www.jimmunol.org/ induced or CD40 ligand-induced up-regulation of these molecules MR/CD206, which belongs to the type I family of C-type lectins, (data not shown). and of the type II lectins DC-ASGPR, DC-SIGN/CD209, and DCs generated in the presence of 10Ϫ6 M SFA from day 0 C1qRP/CD93 was investigated in more detail. Control DC cultures onward demonstrated a slightly decreased T cell stimulatory ca- demonstrated a high expression of MR and DC-SIGN and a mod- pacity (Fig. 3B). Cocultures of SFA-DCs with allogeneic T cells in erate expression of DC-ASGPR and C1qRP, as shown previously a 1:10 ratio resulted in a relative T cell proliferation of 93% (range by others (4, 19Ð21). As observed for the expression of DC-SIGN, from 76% to 112%, p Ͼ 0.05) as compared with coculture with DCs generated in the presence of SFA showed a decreased ex- control DCs. Only cocultures of SFA-DCs and T cells in a 1:300 pression of all lectins tested (Fig. 5A). A detailed dose-response ratio resulted in a significantly decreased T cell proliferation (re- analysis revealed that significant inhibitory effects on the expres- by guest on September 25, 2021 duction: 54 Ϯ 11%, p ϭ 0.0065). sion of MR and DC-SIGN could be observed from 10Ϫ8 M SFA (Fig. 3B). SFA down-regulated the expression of DC-SIGN ϳ100- SFA decreases both lectin-mediated and fluid phase endocytosis fold more efficiently than the expression of MR. We next analyzed whether Ag uptake was influenced by SFA. Less pronounced effects were observed when SFA was added Lectin-mediated endocytosis and fluid phase endocytosis were in- later during the differentiation period. Although SFA decreased the vestigated by analyzing the uptake of dextranFITC or manBSAFITC expression of lectins, of lectin-mediated uptake (Fig. 5C) and of and LY or BSAFITC, respectively. Ligand specificity was con- fluid phase endocytosis (data not shown) when added to already firmed by blocking studies with D-mannose, which demonstrated a differentiated DCs, this reduction was less pronounced compared strong inhibition in the uptake of dextranFITC without changing the with cells exposed to SFA from day 2 onward and especially from uptake via fluid phase endocytosis (Fig. 4A). day 0 onward. DCs cultured in the presence of SFA showed significantly de- RAPA is another immunophilin-binding immunosuppressive creased lectin-mediated endocytosis (reduction: mean 47%; range drug known to inhibit the endocytic capacity of human DCs (8). from 25% to 70%, p ϭ 0.0045) of dextranFITC (Fig. 4B) and man- The presence of RAPA during DC development resulted in cells BSAFITC (data not shown), which was already detected after 5 min with a relatively low expression of MR and a reduced endocytic of incubation and became more clear upon longer incubation time capacity (Fig. 6A). In contrast to SFA, RAPA did not decrease (Fig. 4C). Interestingly, also the fluid phase endocytic capacity was CD1a or DC-SIGN expression, but rather increased the expression significantly decreased by SFA (reduction: mean 46%, range 30% of the latter molecules. Moreover, RAPA strongly induced cell to 62%, p Ͻ 0.0001) (Fig. 4, D and E). Dose-response analysis death (50% reduced cell yield compared with control), which is revealed that SFA inhibited the fluid phase and lectin-mediated also clear from forward/side scatter plots (Fig. 6B) and propidium Ϫ8 Ϫ7 endocytosis to the same extent (IC50,10 Ð10 M)(Fig. 4F). The iodide staining (data not shown) (6). The very low cell viability of inhibitory effects on Ag uptake were not due to a generic toxic cells cultured in the presence of RAPA may contribute to the dif- effect because the incidence of cell death on day 6 of culture was ferences in function and phenotype. not significantly affected by SFA, as determined by trypan blue staining and propidium iodide uptake (Fig. 1B and data not ␥ shown). SFA specifically increases the expression of Fc RII (CD32) and Fc␣RI (CD89) SFA interferes with the induction of C-type lectins during DC Next to C-type lectins, Fc receptors also play an important role in development Ag capture. The expression of the Fc␣R (Fc␣RI/CD89) and the Surface bound C-type lectins are responsible for the uptake of Fc␥R (Fc␥RIII/CD16, Fc␥RII/CD32, Fc␥RI/CD64) were analyzed glycosylated Ags. DC-SIGN is one of a growing number of C-type upon DC generation either in the presence or absence of SFA. The Journal of Immunology 6485

ropinocytosis), as shown by LY uptake, as well as by lectin-mediated endocytosis, as shown by dextranFITC uptake. The decreased uptake of dextranFITC can be explained by the inhibitory effect of SFA on the expression of C-type lectins, such as MR and DC-SIGN. Interestingly, SFA increased the expression of the Fc receptors CD89 and CD32. Both the inhibitory as well as the stimulatory effects of SFA were dose-dependent and already observed with 10Ϫ8 M. As immature cells, scattered throughout the body, DCs have the capacity to recognize and take up a wide range of self and non-self Ags. DCs use different mechanisms, including ﬂuid phase and receptor-mediated endocytosis, for the uptake of Ags, such as C-type lectin and Fc receptor-mediated uptake. DCs discriminate self from non-self using conserved pattern-recognition receptors, which recognize molecular patterns at the cell surface of all mi- croorganisms. Receptors of this type include Toll-like receptors (22, 23) and C-type lectins (24). Some of the C-type lectins are shared with other cell types, such as the MR, whereas other receptors are more DC restricted, e.g., DEC205/CD205, which has Downloaded from very low expression on human blood DCs; Langerhans cells and in vitro-cultured DCs; Langerin/CD207, which is exclusively expressed on Langerhans cells; DC-SIGN/CD209 expressed on interstitial DCs; and a small subset of blood DCs, including an AS- GPR expressed on interstitial DCs and BDCA-2 on plasmacytoid DCs (19, 25). http://www.jimmunol.org/ SFA is the ﬁrst immunosuppressive drug that shows inhibitory effects on the expression of DC-SIGN. DC-SIGN is an adhesion receptor important for several DC functions (26). DC-SIGN can interact with the carbohydrate-bearing self-glycoproteins ICAM-2 and ICAMÐ3 to mediate chemokine-induced transmigration of DCs across endothelium (27) and transient adhesion between DCs and T cells enabling complete TCR engagement (21), respectively.

In addition, C-type lectins like DC-SIGN recognize pathogens by guest on September 25, 2021 through their carbohydrate profiles and internalize the pathogen for Ag processing and presentation (25, 28, 29). Detailed knowledge about pathogenic targets as well as cellular ligands, including the identity of the carbohydrate structure they recognize, is lacking for most of these receptors. Concerning pathogen-recognition, DC- SIGN is involved in binding of HIV-1, CMV, and Ebola virus resulting in enhanced cis and trans infection (21, 30, 31). Recently, FIGURE 3. Influence of SFA on T cell stimulatory capacity of DCs in it was found that DC-SIGN also recognizes nonviral pathogens, allogeneic MLR. A, Addition of different concentrations of SFA to an including Mycobacterium tuberculosis, Helicobacter pylori, Leish- allogeneic MLR with immature control-DCs derived from two different mania mexicana, and Schistosoma mansoni (32, 33). It appears Ϯ donors is shown. T cell proliferation is expressed as mean cpm SD of that viral pathogens target DC-SIGN for transmission, whereas triplicate cultures. B, Day 6 with control DCs and SFA DCs (10Ϫ6 M) were nonviral pathogens modulate DC-induced immune activation (34). harvested, extensively washed, and used in an allogeneic MLR. T cell proliferation is expressed as mean cpm Ϯ SEM of five experiments with Next to DC-SIGN, the MR has been shown to recognize myco- different donors. bacteria and fungi/protozoa (35), and probably also other C-type lectin-pathogen interactions will be discovered in the future. This means that an altered expression of C-type lectins such as DC- SIGN and MR may have profound effects on the infection efficiency Control DCs showed expression of CD32 and CD89 surface mol- and subsequent immune response to these types of pathogens. ecules, but did not express CD16 and CD64 (Fig. 7A), as previ- Next to C-type lectins, DCs use Fc receptors to efficiently in- ously demonstrated (9). DCs generated in the presence of SFA ternalize Ag. Fc receptors evolved to bind the Fc fragment of Igs were also negative for CD16 and CD64, but demonstrated an in- for uptake of Ags opsonized with specific Abs. IgG and IgA, creased expression of CD32 and CD89. CD32 and CD89 were which bind to Fc␥R and Fc␣R, respectively, are the most abundant up-regulated to the same extend and in a dose-dependent manner, Ig isotypes present in human blood. Two types of Fc␥Rs exist: starting at a concentration of 10Ϫ8 M SFA (Fig. 7). activating and inhibitory Fc␥R. Activating Fc␥Rs include Fc␥RI, Fc␥RIIb, and Fc␥RIII (36). Fc␥RIIa, which does not exist in mice Discussion but does exist in human, inhibits cell activation through immuno- Modulation of T lymphocyte function by SFA is well documented. receptor tyrosine-based inhibitory motifs. Human immature mono- The present study demonstrates that SFA also interferes with DC cyte- and CD34-derived DCs only express Fc␥RII/CD32 (9, 37, function. SFA strongly decreases the efficacy of Ag uptake by DCs 38)(Fig. 6), but the relative expression of activating and inhibitory through at least two means: by fluid phase endocytosis (or mac- isoforms of CD32 is not known. Ligation of Fc␥R is known to 6486 SFA SPECIFICALLY AFFECTS ENDOCYTOSIS BY HUMAN DC

FIGURE 4. SFA decreases both lectin-mediated and fluid phase endocytosis. A, DCs were generated under control conditions. Lectin-mediated (dextranFITC) and fluid phase LY en- Downloaded from docytosis were determined after 30 min of incubation either in the presence or absence of 50 mM D-mannose. Data shown are representative of three independent experiments. BÐE, DCs were generated under control conditions, in the presence of http://www.jimmunol.org/ SFA (10Ϫ6 M) or in the presence of methanol (1/10000). Uptake of either dextranFITC (B and C)orLY(D and E) was evaluated after 30 min (B and D) or at different time points during a 60-min incubation period (C and E). Negative controls were incubated with the respective Ag at 4¡C and showed similar mean fluorescence in- by guest on September 25, 2021 tensities. Data shown are representative of four independent experiments. F, DCs were generated under control conditions or in the presence of SFA (10Ϫ9Ð10Ϫ6 M). Data shown are the mean (Ϯ SEM) relative uptake of either dextranFITC or LY after 30 min of incubation compared with the uptake of the compounds by control DC of three independent experiments with different donors.

induce the maturation of mouse DCs, whereas targeting Fc␥Rs on manner (38), making these DCs rather tolerogenic than immuno- human DCs only partially triggers DC maturation (38, 39). IgG- genic (40, 41). induced semimature human DCs do not produce IL-12, but dem- The best characterized receptor for IgA in humans is Fc␣RI/ onstrated an increased production of IL-10 in a CD32-dependent CD89. Both in vivo and in vitro, it has been shown that CD89 is The Journal of Immunology 6487 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. The effect of SFA on the expression of C-type lectins. A, DCs were generated under control conditions or in the presence of SFA (10Ϫ6 M) or methanol. Cell surface expression of the indicated C-type lectins was analyzed by flow cytometry as described in Materials and Methods. Data shown are representative of five independent experiments. B, DCs were generated in the presence of different concentrations of SFA. On day 6, cells were analyzed for their expression of MR and DC-SIGN by flow cytometry. Data shown are the mean (Ϯ SEM) of relative expressions of MR or DC-SIGN compared with the expression on control DC of three independent experiments with different donors. C, Monocytes were cultured for 7 days with IL-4 and GM-CSF. SFA (10Ϫ6 M) was added to the cultures on day 0, day 2, or day 6. Cell surface expression of the indicated proteins and uptake of dextranFITC was analyzed by flow cytometry as described in Materials and Methods. Experiment was performed in duplicate and is representative of three independent experiments. expressed on interstitial DCs, but not Langerhans cells (9, 42, 43). The molecular mechanism by which SFA exerts its immuno- As was found for CD32 cross-linking on DCs, CD89 triggering modulatory effects remains to be elucidated. With regard to the with IgA immune complexes resulted in rapid internalization and effects of SFA on DC function, it seems that SFA is more closely subsequent semimaturation of DCs, characterized by a partially related to RAPA than to CsA, as found for its effect on T cells (14, increased CD86 expression and high IL-10 production (42). 15). Calcineurin-inhibition with either FK506 or the cyclophilin-A 6488 SFA SPECIFICALLY AFFECTS ENDOCYTOSIS BY HUMAN DC

FIGURE 6. RAPA affects cell phenotype, function, and viability. DCs were generated under control conditions or in the presence of either SFA (10Ϫ6 M) or RAPA (10Ϫ6 M). Cell surface expression of the indicated proteins and uptake of dextranFITC (A) were analyzed by ﬂow cytometry as described in Materials and Methods. Cells were gated on forward/side scatter (B) and propidium iodide-positive cells were excluded from analysis. Exper- iment was performed in duplicate and is representative of two independent experiments. Downloaded from http://www.jimmunol.org/

binding drug CsA affected neither Ag uptake receptor expression nor the endocytic activity of DCs (8Ð10)(data not shown). In contrast, we observed that RAPA, in accordance with previous reports (8), decreased the expression of MR and also reduced ﬂuid phase (data not shown) and lectin-mediated endocytosis as was found for SFA. However, RAPA also dramatically induced cell death (6), which may account for the decreased functional capacity of the

cells. SFA increased the expression of CD32, an effect that has not by guest on September 25, 2021 been observed with RAPA (8). Delayed exposure of developing DCs to SFA showed less pronounced effects on DC function and phenotype. This observation may explain the apparent discrepancy between the present data and the recent study of Steinschulte et al. (16) in which it was demonstrated that CD1a expression was not significantly affected when SFA was added on day 2 of DC differentiation. The latter study did not comment on Ag receptor expression or endocytic capacity. Furthermore, both RAPA (our un- published observation and Ref. 8) and SFA are very active when present during DC development, but when added to already differentiated immature DCs the effects on phenotype and endocytosis are less and sometimes even absent. These data suggest that SFA influences the development of the endocytic machinery by targeting a signaling molecule already present in monocytes. In view of the effect on endocytosis and the decreased ability to produce IL-12 (16) it is most likely that this target remains present in differentiated DCs. Controversial data exist about the effect of CsA on the T cell stimulatory capacity of DCs. It seems that both CsA (11) and SFA, if so, only moderately decrease their ability to stimulate T cells. It has been suggested that RAPA partially reduces the T cell stimu- FIGURE 7. SFA dose dependently increases the expression of Fc␥RII latory capacity of human DCs in allogeneic MLRs (8), but the and Fc␣RI. A, DCs were generated under control conditions or in the observed reduction in T cell proliferation may be explained by a Ϫ6 presence of SFA (10 M). Cell surface expression of the indicated Fc decreased DC viability due to RAPA-induced apoptosis (6, 8). The receptors was analyzed by flow cytometry as described in Materials and tendency of a decreased potency of SFA-DCs to activate T cells Methods. Data shown are representative for one of five independent experi- can be the consequence of a decreased expression of DC-SIGN, as ments. B, DCs were generated under control conditions or in the presence of SFA (10Ϫ9Ð10Ϫ6 M). On day 6, cells were analyzed for their expression of adhesion molecules are important in DC-T cell interactions (21), CD32 (Fc␥RII) and CD89 (Fc␣RI) by flow cytometry. Shown are the relative and/or the consequence of a decreased production of growth fac- expressions of CD32 and CD89 compared with the expression on control DC tors such as observed for IL-12 (16). However, also inevitable of two independent experiments with different donors. carry over effects of the drug cannot be excluded. The role of CD1 The Journal of Immunology 6489 molecules on DCs remains largely unknown. Nevertheless, it is not 19. Valladeau, J., V. Duvert-Frances, J. J. Pin, M. J. Kleijmeer, S. Ait-Yahia, expected that CD1a plays a major role in the stimulation of allo- O. Ravel, C. 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