Journal of Drug Targeting, 2003 Vol. 11 (1), pp. 11–18
Improved Stimulation of Human Dendritic Cells by Receptor Engagement with Surface-modified Microparticles ¨ RO ¨ Sb, MARKUS TEXTORb, MARTINA KEMPFa, BARNALI MANDALa, SAMANTHA JILEKa, LARS THIELEa, JANOS VO HANS P. MERKLEa and ELKE WALTERa,* a
Department of Applied BioSciences, Drug Formulation & Delivery Group, Swiss Federal Institute of Technology Zurich (ETH), Winterthurerstrasse 190, CH-8057 Zurich, Switzerland; bLaboratory of Surface Science and Technology, Department of Materials, Swiss Federal Institute of Technology Zurich (ETH), Wagistr. 2, 8952 Schlieren, Switzerland (Received 2 November 2002)
Dendritic cells (DC) need to be stimulated before they can function to initiate immune responses. This study investigates whether microparticles loaded with antibodies specific for selected receptors expressed by DC can induce stimulation of these cells. Plain microparticles were compared with microparticles which were surface-loaded with specific antibodies for human CD40, Fcg, avb3 and avb5 integrin receptors. The antibodies were either physically adsorbed or covalently linked to the microparticle surface. Anti-CD40 antibody and human IgG immobilised on the surface of microparticles induced enhanced DC maturation and activation as expressed by CD83 and CD86 upregulation. IL-12 secretion was induced at a detectable but relatively low level. Both anti-integrin antibodies (anti-avb3 and anti-avb5) induced comparable and considerable maturation of DC, but only anti-avb3 antibody induced significant activation of DC, whereas anti-avb5 did not. The stimulatory effects were most pronounced by employing microparticles with covalently linked antibodies, but were also observed to a minor extent when the antibodies were physically adsorbed to polystyrene and biodegradable poly(lactide-co-glycolide) microparticles. Engineering of microparticles by surface conjugation of specific ligands to stimulate DC may increase the effectiveness of microparticulate vaccine delivery systems. Keywords: Dendritic cells; Microparticles; Stimulation; Specific antibodies; Surface loading
INTRODUCTION Dendritic cells (DC) play a key role in the induction of acquired and innate immunity (Banchereau et al., 2000; Foged et al., 2002). They represent professional antigen presenting cells and are able to capture antigens, whole microbes, viruses, apoptotic bodies and to a certain extent, biodegradable microparticles (Inaba et al., 1993; Albert et al., 2000; Thiele et al., 2001; Walter et al., 2001). The function of DC is highly influenced by their level of maturation. Upon stimulation by recognition of characteristic patterns of pathogens as well as by inflammatory cytokines and necrotic cells, DC undergo a transition from the immature to the mature state. At an immature state, DC act as sentinels in the peripheral tissues, continuously sampling antigenic material at their site of entry. During maturation, DC undergo phenotypic and functional changes that can culminate in the complete transition of immature antigen-capturing cells to mature activated
antigen-presenting cells providing additional signals or much stronger versions of the same signal (activated DC). Surface markers, such as CD83 and CD86, become upregulated and secretion of immunostimulatory cytokines, such as IL-12, is induced (Banchereau et al., 2000; Langenkamp et al., 2000). Recently, additional signals have been identified that stimulate DC maturation, such as CD40 cross-linking (Caux et al., 1994; Osada et al., 2002) and stimulation of the Fcg-receptor (Regnault et al., 1999). On the contrary, receptors responsible for the internalization of apoptotic cells, such as the avb5 integrin receptor expressed by DC, may inhibit immunostimulatory signals in order to not respond to self-antigens (Albert et al., 2000; Green and Beere, 2000). Microparticles are valuable carriers for the delivery of antigens because they are naturally targeted to DC by unspecific phagocytosis as demonstrated in vitro (Thiele et al., 2001; Walter et al., 2001) and in vivo (Lunsford et al., 2000; Newman et al., 2002). In some cases,
*Corresponding author. Tel.: þ 41-76-5738777. Fax: þ41-1-6356881. E-mail:
[email protected] ISSN 1061-186X print/ISSN 1029-2330 online q 2003 Taylor & Francis Ltd DOI: 10.1080/1061186031000072978
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biodegradable microparticles have even been shown to mediate sufficient immune responses when the antigen is concentrated in the microparticle and protected against premature proteolytic degradation (Men et al., 1999; Johansen et al., 2000). Thus, microparticles are multifunctional that they efficiently deliver antigens to DC and simultaneously act as an adjuvant. Moreover, microparticles can be modified by incorporation or physical association of immunostimulatory agents in order to enhance DC stimulation (Singh et al., 2001; Jilek et al., 2002). In this study, we investigated the effect of microparticles on the stimulation of human monocyte-derived DC. We compared plain microparticles with microparticles which were surface-loaded with specific antibodies for human CD40, Fcg, avb3 and avb5 integrin receptors. The antibodies were either physically adsorbed or covalently linked to the microparticle surface. Maturation and stimulation of DC was assessed by the expression of specific surface markers and the secretion of IL-12. Our results demonstrate that specific antibodies loaded onto the surface of microparticles stimulate the maturation and the secretion of immunostimulatory cytokines in human DC. As a proof-of-concept our studies further envisage the potential of tailored particulates as vaccine delivery systems carrying specific signals for DC maturation and activation on their surface, in addition to the embodied antigen.
MATERIAL AND METHODS Materials Carboxylated polystyrene particles of 4.5 mm in diameter were obtained from Polysciences Europe, Germany. Particles were washed three times with sterile endotoxin-free Hepes buffer (10 mM, pH 7.4) prior to use. Poly(D ,L -lactide-co-glycolic acid) (PLGA) type polymer (Resomer RG502H, ratio lactide: glycolide 50:50, MW 14,000, uncapped end groups) was obtained from Boehringer Ingelheim (Ingelheim, Germany). Material for cell cultures was purchased from Life Technologies AG (Basel, Switzerland). Lipopolysaccharide was obtained from Sigma (LPS; Escherichia Coli 055:B5). All other chemicals used were of analytical grade unless otherwise specified and obtained by Sigma (Buchs, Switzerland). PLGA Microparticles PLGA microparticles were prepared by spray drying as previously described (Walter et al., 1999). Briefly, PLGA polymer was dissolved in dichloromethane (w/w, 5%) and was spray-dried in a laboratory spray-dryer (Model 190, Bu¨chi, Switzerland) at an inlet temperature of 458C. The spray-flow was set at 600 nl/h and the product feed was 3 ml/min. The microparticles were washed with water,
collected on a 0.45 mm cellulose acetate membrane filter and dried under vacuum for 24 h. The microparticles were stored at 48C. Size distribution of the microparticles was measured by laser light scattering (Mastersizer X, Malvern Instruments Ltd., Worcestershire, UK; equipped with a 45 mm lens). Calculation of particles sizes was based on Mie’s theory. The particle size distribution was presented in the volume-weighted mode and revealed particles in the range of 1– 10 mm as previously described in detail (Walter et al., 1999; 2001). The total surface area of the particles was calculated from the particle size distribution after assessing particle density by using a gas displacement pycnometer (AccuPyc 1330, Micromeritics, Belgium). Endotoxin Test Microparticles were dispersed (1 mg/ml) in endotoxin-free water and incubated at room temperature under gentle end-to-end shaking. The aqueous phase was then tested for the presence of endotoxin using the QCL-1000w Chromogenic Limulus Amebocyte Lysate (LAL) kit (Biowhittaker, Verviers, Belgium). Surface-loading of Microparticles with Antibodies Polystyrene particles were covalently coated with various antibodies and protein (anti-human CD40, antihCD40/TNFSF5, R&D Systems, Wiesbaden-Nordenstadt, Germany; anti-human avb3, MAB 19767, Chemicon, Lucerne, Switzerland; anti-human avb5, MAB 20197, Chemicon; human IgG, Sigma; bovine serum albumin (BSA), Sigma) by conjugation to the free carboxyl groups on the particle surface using the carbodiimide method (data sheet #238 C, Polysciences, Inc.). Physical adsorption to the surfaces of polystyrene particles and PLGA microparticles was performed in endotoxin-free Hepes buffer (10 mM, pH 7.4) by using a nominal loading of 100 mg/mg of microparticles. Microparticles were incubated at room temperature under gentle end-to-end shaking for 15 min and subsequently centrifuged (5 min, 5000 rpm, Eppendorf, Centrifuge 5417R, EppendorfNetheler-Hinz GmbH, Germany) to remove excess of antibody. Redispersion was performed in Hepes buffer by slightly vortexing. The amount of unbound antibody was determined in the supernatants upon conjugation with fluorescamine (0.3 mg/ml in acetone) according to Lorenzen and Kennedy (1993). Quantitative analysis was performed by using a fluorescence-photometer (Cary Eclipse, Varian, Australia; excitation 360 nm/emission 480 nm). Surface-loading of Glass Coverslips with Antibodies Prior to loading, the glass coverslips (12 mm in diameter) were oxygen-plasma cleaned in a Plasma Cleaner/Sterilizer PDC-32G instrument (Harrick, USA) for 7 min. Loading was performed in 24-well plates under aseptic
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conditions with 5 mg of the respective antibodies dissolved in 100 ml 10 mM Hepes buffer pH 7.4 for 15 min at room temperature. Control coverslips were incubated in Hepes buffer only. After coating, the coverslips were rinsed twice for 5 min with PBS and once with RPMI 1640 medium prior to adding the cells. Release of Surface-bound Antibody from Microparticles and Coverslips The release of surface-bound antibody in the presence of cell culture medium over time was tested by employing a fluorescence-labelled model antibody (human FITC-IgG, Sigma). Surface-loading of microparticles and glass coverslips was performed as described above and the loading was calculated from the amount of unbound antibody in the supernatants. Microparticles were subsequently dispersed in cell culture medium (poylstyrene: 107 particles per 6-well, PLGA: 300 mg per 6-well) and glass coverslips were placed in 24-well plates in the presence of cell culture medium. All samples were incubated for several time intervals at 378C and the release of antibody was determined in the supernatant. Quantitative analysis of FITC-IgG was performed by using a fluorescence-photometer (Cary Eclipse, Varian, Australia; excitation 480 nm/emission 520 nm). DC Cell Cultures DC were obtained from human peripheral blood according to Sallusto et al. (1995). Briefly, peripheral blood monocytes obtained from buffy coats (Blood-bank Zurich, Switzerland) were isolated by density gradient centrifugation on Ficoll-Paque (Pharmacia Biotech, Dubendorf, Switzerland). Peripheral blood monocytes were resuspended in RPMI 1640 supplemented with 10% heat inactivated (pooled) human serum (Bloodbank Zu¨rich, Switzerland) and then allowed to adhere for 2 h in 6-well plates (600,000 cells per well). Non-adherent cells were removed and adherent cells were further cultured in RPMI 1640 supplemented with 5% heat inactivated (pooled) human serum in the presence of 1000 IU/ml IL-4 (Sigma, Switzerland) and 50 ng/ml GM-CSF (R þ D Systems). Cultures were kept at 378C in 5% CO2 humidified atmosphere.
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without any additions, or by adding the maturation inducer lipopolysaccharide (LPS) (1 mg/ml). After 24 h, DC were analysed for CD83 and CD86 surface expression and the cell culture medium was checked for IL-12 secretion. Assessment of DC Stimulation upon Exposure to Surface-loaded Coverslips DC incubated for one week in 6-well plates were added to 24-well plates with each well (200,000 cells in 1 ml cell medium) containing either antibody-loaded or plain control coverslips. Additionally, LPS-stimulation was performed in the presence of plain control coverslips. After 24 h, DC were analysed for CD83 and CD86 surface expression and the cell culture medium was checked for IL-12 secretion. Analysis of Surface Marker Expression Surface marker expression of DC was analysed by flow cytometry. DC were recovered from the 6-well plates and washed once with plain medium. The DC were then incubated (45 min, 48C) with the following primary antihuman antibodies: CD83 (Clone HB15e, Pharmingen, Lucerne, Switzerland) and CD86 (Clone IT2.2, Pharmingen). Control cells were processed similarly with mouse isotype control antibodies IgG1 (MOPC-21, Sigma). Cells were washed once and subsequently incubated with the secondary antibody (anti-mouse IgG R-Phycoerythrin conjugate, Sigma) for 45 min at 48C, washed twice and transferred to FACS tubes (Falcon, Becton Dickinson AG, Basel, Switzerland) for analysis by flow cytometry (FACScan, Becton Dickinson). According to Cochand et al. (1999), more than 90% of the cells were identified as DC. Analysis of Cytokine Secretion The cell culture medium from the DC stimulation experiments was harvested and tested for the presence of IL-12(p40) using an enzyme linked-immunosorbent assay (ELISA; Pharmingen).
RESULTS
Assessment of DC Stimulation upon Addition of Surface-loaded Microparticles
Characterization of Antibody Binding to Microparticles and Glass Surfaces
At day 7 or 8 of culture, DC were incubated with 107 of polystyrene particles or 300 mg of PLGA microparticles in 3 ml of cell culture medium in 6-well plates. When incubated with polystyrene and PLGA microparticles, cell cultured DC dispersions show substantial phagocytic uptake of the microparticles. Full experimental protocols were previously established by our group (Thiele et al., 2001; Walter et al., 2001) and published together with associated data on phagocytosis. Controls were performed
The loading of antibody onto microparticles and flat glass surfaces was performed by either covalent conjugation or physical adsorption. Moreover, the persistence of the antibody on the respective surfaces was tested in the presence of serum-containing medium over time. For this purpose, a fluorescent-labelled anti-human IgG antibody was employed in this study. Plasma-cleaned flat glass surfaces were readily loaded with antibody by physical adsorption with the major fraction of the antibody
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FIGURE 1 Release of FITC-IgG model antibody from the surface of glass coverslips (glass) or polystyrene (PS) and PLGA microparticles. The antibody was either physically adsorbed (ad) or covalently linked (co) upon incubation in serum-containing cell culture medium. The initial loading is expressed as ng/mm2 and is designated in parentheses. Error bars represent standard deviations of n ¼ 3:
remaining on the surface over a period of 24 h (Fig. 1). In contrast, over 50% of the antibody adsorbed to polystyrene particles was released into the incubation medium already after a short period of time (Fig. 1). Moreover, antibody adsorbed to biodegradable PLGA microparticles was completely desorbed from the particle surface after 4 h. In order to provide high antibody loading on the microparticle surface during the stimulation experiments, we covalently conjugated the antibody to the surface of carboxylated polystyrene. Thus, covalent conjugation revealed a higher surface loading compared to plain adsorption as indicated in Fig. 1. Moreover, the total amount of covalently-linked antibody remained on the particle surface for 24 h (Fig. 1). Up-regulation of Surface Markers in DC upon Exposure to Antibody-loaded Microparticles or Glass Surfaces Various antibodies specific for selected receptors present on human DC were loaded onto either microparticles or flat glass surfaces. We tested the influence of antibodyloading on the stimulation of DC by measuring the surface expression of CD83 and CD86. CD83 is a relevant marker for DC maturation and is exclusively expressed on mature DC (Zhou and Tedder, 1996), whereas CD86 is present on immature and mature DC and is overexpressed upon DC activation (Banchereau et al., 2000; Shortman and Liu, 2002). Thus, the samples were analysed with regard to the percentage of CD83 positive cells and the relative mean fluorescence of CD86 positive cells. As controls, nontreated DC and LPS-stimulated DC were used. The results are expressed as percent in relation to LPS-treated and non-treated cells in order to eliminate the variations of the different donors (Langenkamp et al., 2000). In order to eliminate the effect of LPS contamination, if any, on the stimulation of DC, we checked all buffers and
FIGURE 2 Expression of the maturation marker CD83 upon exposure to antibody-loaded surfaces to DC. Various antibodies were physically adsorbed (ad) to flat plasma-cleaned glass surfaces (glass) or to polystyrene (PS) and PLGA microparticles. Additionally, covalent linkage to polystyrene particles (co) was performed. Error bars represent standard deviations ðn ¼ 3 – 5Þ; except for experiments involving PLGA microparticles or avb3/avb5 antibodies where error bars represent the range between two experimental data sets ðn ¼ 2Þ:
surfaces for the presence of endotoxins by employing the LAL assay. No detectable LPS was found on glass surfaces and in the incubation buffers. The presence of LPS on the microparticle surfaces was found to be 3000 times less than the amount of LPS needed for the stimulation of the DC. Exposure of DC to antibody-loaded surfaces resulted in a considerable up-regulation of CD83 expression compared to plain surfaces or the addition of soluble antibody (Fig. 2). Adsorbed anti-CD40 appeared to have the most pronounced effect which was higher than antiIgG for flat surfaces, polystyrene particles and PLGA microparticles. Covalent coating to the particle surface considerably enhanced the stimulatory effect of the antibodies, which may be due to the higher loading or prolonged binding in the presence of serum-containing medium (Fig. 1). Particles coated with bovine serum albumin or particles treated as in the coating procedure without adding any protein for conjugation did not enhance the up-regulation of CD83 and CD86 compared to plain particles (data not shown). This indicates that the chemical modification of the particle surfaces which occurs during the covalent linkage procedure did not negatively affect the stimulation of DC. In addition to covalently linked human IgG and anti-CD40, anti-avb3 and anti-avb5 induced a comparable up-regulation of CD83. The data on the up-regulation of CD86 are generally in accordance with those of CD83. Here, especially surfacebound human IgG resulted in an noticeable effect compared to plain surfaces of microparticles and flat glass surfaces (Fig. 3). Moreover, covalently-linked antiavb3 induced high stimulation of DC which exceed those induced by anti-avb5. The addition of comparable amounts of soluble anti-avb3 or anti-avb5 did not result in any noticeable up-regulation of CD83 or CD86 (data not shown).
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production was enhanced when polystyrene particles were loaded with the various antibodies and was most pronounced with covalently linked antibody as compared to physical adsorption. The addition of comparable amounts of soluble antibodies did not result in any noticeable secretion of IL-12 (Fig. 4, data not shown). Interestingly, unloaded PLGA microparticles already induced comparatively high amounts of IL-12 which was not further enhanced by adsorption of anti-CD40 or human IgG antibody.
FIGURE 3 Expression of the activation marker CD86 upon exposure to antibody-loaded surfaces to DC. Various antibodies were physically adsorbed (ad) to flat plasma-cleaned glass surfaces (glass) or to polystyrene (PS) or PLGA microparticles. Additionally, covalent linkage to polystyrene particles (co) was performed. Error bars represent standard deviations ðn ¼ 3 – 5Þ; except for experiments involving PLGA microparticles or avb3/avb5 antibodies where error bars represent the range between two experimental data sets ðn ¼ 2Þ:
Secretion of IL-12 by DC upon Exposure to Antibody-loaded Microparticles IL-12 is an important regulator of cell-mediated immune responses and is produced by mature and stimulated DC (Macatonia et al., 1995; de Saint-Vis et al., 1998). The bioactive molecule IL-12(p70) is a heterodimer formed by two subunits (the p35 and the p40 subunit) (Carra et al., 2000). IL-12(p40) has been reported to be selectively produced by activated APC (Takeshita and Klinman, 2000) and is secreted at a 100- to 1000-fold excess over the bioactive IL-12(p70) (Carra et al., 2000). Since antibody-loaded microparticles were able to stimulate the maturation of DC, we checked whether in addition, the secretion of IL-12(p40) was induced. DC were exposed to various antibody-loaded microparticles for 24 h and the culture medium was tested for secreted IL-12(p40) by ELISA. Secretion of IL-12 was in good accordance with data on CD83 up-regulation, but was comparatively low in relation to LPS-stimulated cells (Fig. 4). Generally, IL-12
FIGURE 4 Secretion of IL-12 upon exposure to antibody-loaded microparticles to DC. Various antibodies were physically adsorbed (ad) to polystyrene (PS) or PLGA microparticles. Additionally, covalent linkage to polystyrene particles (co) was performed. Error bars represent standard deviations ðn ¼ 3 – 5Þ; except for experiments involving PLGA microparticles or avb3/avb5 antibodies where error bars represent the range between two experimental data sets ðn ¼ 2Þ:
DISCUSSION DC need to be stimulated before they can function to initiate immune responses. In addition to characteristic signals from pathogens and inflammatory cytokines, selected receptor-mediated stimulation has been reported to lead to the maturation and activation of DC (Caux et al., 1994; Regnault et al., 1999; Osada et al., 2002). Antigenloaded microparticles have been demonstrated to be able to efficiently deliver antigen to DC (Lunsford et al., 2000; Thiele et al., 2001; Walter et al., 2001; Newman et al., 2002) and even enhance antigen presentation to T cells compared to soluble antigen (Shen et al., 1997; Svensson et al., 1997). In vivo studies revealed noticeable immune stimulation indicating a potential adjuvant effect of the microparticle formulations (Johansen et al., 2000). Thus, the aim of this study was to investigate whether microparticles which were surface-loaded with specific antibodies for selected receptors expressed by DC, can induce stimulation of these cells. We found that antibodyloaded microparticles considerably induce maturation and activation of DC as indicated by the up-regulation of the surface markers CD83 and CD86 and the secretion of IL-12. Thus, we demonstrated that tailored microparticles may provide a potential antigen delivery systems by simultaneously exhibiting stimulatory adjuvant-type properties. CD40 receptor is expressed on DC and is essential for the activation of these cells and for the generation of cytotoxic T cells (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998). The interaction of receptorspecific ligand with CD40 is suggested to condition the DC and enhance their capacity to present antigens to cytotoxic T cells by increased expression of costimulatory molecules and upregulation of cytokine production (Caux et al., 1994; Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998; Cella et al., 2000). We found that surface-bound CD40 antibody was able to induce the up-regulation of the maturation marker CD83, whereas comparable amounts of soluble CD40 in the incubation medium did not show a detectable effect. Our data are in accordance to findings by others demonstrating that anti-CD40 antibody immobilised on cell culture plates resulted in DC expressing higher levels of CD83 than those supplemented with an equivalent quantity of this antibody dissolved in
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the medium (Osada et al., 2002). CD40-matured DC showed higher stimulation of naive allogenic T cells than DC without CD40 stimulation (Cella et al., 2000; Osada et al., 2002). In our study, the effect on DC maturation was most pronounced when the antibody was covalently linked to the surface of model polystyrene particles. This is attributed to the functional exposure of a high concentration of covalently linked antibody on the particle surface which is fully maintained during incubation in serum-containing medium, thus enabling efficient crosslinking with the CD40 receptor on DC. Moreover, antibody-loaded microparticles displayed a generally higher capacity in inducing DC maturation compared to antibody adsorbed to flat glass surfaces. Thus, microparticles loaded with specific ligands for the CD40 receptor may be particularly useful in eliciting mature DC in vivo. IgG serves as a ligand for the Fcg-receptor, and Fcgreceptor engagement has been reported to promote DC maturation (Regnault et al., 1999). In this study, surfacebound human IgG resulted in only slightly enhanced up-regulation of CD83 as compared to unloaded microparticles. This could be accounted to the fact that IgG is already present in the incubation medium and may adsorb to unloaded microparticles. Substantial adsorption of IgG to polystyrene and PLGA microparticles from serum has indeed been demonstrated in previous studies (Muller et al., 1997; Luck et al., 1998). In contrast, covalently bound IgG induced noticeably enhanced up-regulation of CD83, again indicating the importance to maintain a functional exposure combined with high concentration of ligand on the particle surface. In vivo studies employing microparticles loaded with human IgG revealed enhanced local and systemic immune responses against xenotypic epitopes after instillation into the respiratory tract of mice (Bot et al., 2000). The high local concentration of IgG provided by the microparticle formulation is suggested to enhance immunostimulation upon interaction with the Fcg-receptor of professional antigen-presenting cells. Activation of DC as detected by the up-regulation of the co-stimulatory molecule CD86 was in good accordance with data on the up-regulation of CD83. Especially, covalently linked anti-CD40 antibody and human IgG induced noticeable activation of DC compared to unloaded polystyrene particles. The engagement of both receptors has been previously reported to induce full DC activation, reflected by increased expression levels of major histocompatibility complexes and co-stimulatory molecules at the cell surface (Caux et al., 1994; Regnault et al., 1999; Cella et al., 2000). Interestingly, no significant effect on the up-regulation of CD86 was observed with anti-CD40 antibody immobilised on cell culture plates. This is in accordance with our findings when anti-CD40 antibody was adsorbed to flat glass surfaces and may be accounted to the lower concentration of antibody compared to covalently linked antibody on the surface of polystyrene particles.
IL-12, which is produced by mature DC plays a vital role in the development of T helper cell type 1 mediated cellular responses. Exposure to antibody-loaded microparticles induced only low amounts of secreted IL-12 as compared to LPS-stimulated DC. The effect of unloaded microparticles may again be accounted to human IgG which is likely to be adsorbed from the incubation medium to the particle surface. Covalent loading of antiCD40 antibody and human IgG appeared to slightly increase IL-12 secretion compared to soluble antibody or unloaded polystyrene microparticles. According to previous reports, CD40 engagement alone does not induce high-level production of IL-12 in DC, but requires additional IL-12 inducers, such as interferon-g (Hilkens et al., 1997; Kalinski et al., 1999; Osada et al., 2002). Thus, the effect of microparticle loaded anti-CD40 antibody needs to be further evaluated in the presence of additional IL-12 inducers. The effect of plain PLGA microparticles on IL-12 secretion is quite surprising and can only be speculated on up to date. A detailed study on the effect of various types of PLGA and poly(D ,L lactide) microparticles is currently ongoing in our group. Preliminary results indicate that only fast swelling PLGA polymer carrying uncapped end groups as used in this study induced significant secretion of IL-12 as compared to others (unpublished results). The fast swelling of the polymer is thought to trigger a stimulus inside the cells which may lead to enhanced stimulation of DC. Previous coating of PLGA microparticles with antibodies is performed in an aqueous environment and may lead to preliminary swelling already before entering the cells and may thus diminish the stimulation of IL-12 secretion. Both anti-integrin antibodies (anti-avb3 and antiavb5) induced comparable and considerable maturation of DC combined with relatively low IL-12 secretion when covalently linked to the surface of polystyrene particles. Interestingly, only anti-avb3 antibody induced significant activation of DC, whereas anti-avb5 did not. These data are in accordance with the physiological function of the receptors. Phagocytosis of apoptotic cells by DC has been demonstrated to occur via the avb5 integrin receptor (Albert et al., 2000), but does not induce the upregulation of co-stimulatory molecules (Gallucci et al., 1999). Presentation by DC in the lymph node in this case is suggested to lead to the induction of T cell tolerance (Gallucci et al., 1999; Shortman and Liu, 2002). The avb3 integrin receptor, on the other hand, has been reported to be involved in the migration of monocytes and in the uptake of apoptotic cells by macrophages (Weerasinghe et al., 1998; Finnemann and Rodriguez-Boulan, 1999), whereas its function in DC has not yet been clarified. In summary, we found that receptor engagement by employing antibody-loaded microparticles considerably enhanced the stimulation of DC. Covalently linked anti-CD40 antibody and human IgG on the surface of polystyrene microparticles induced enhanced DC maturation and activation as expressed by CD83 and CD86
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up-regulation. IL-12 secretion was induced at a detectable but relatively low level and may need additionally inducers, such as interferon-g (Hilkens et al., 1997; Kalinski et al., 1999; Osada et al., 2002) or immunostimulatory DNA sequences (Sparwasser et al., 2000). The stimulatory effects were most pronounced upon exposure to microparticles with covalently linked antibodies, but were also observed to a minor extent when the antibodies were only physically adsorbed to polystyrene and PLGA microparticles. Thus, our work suggests as a proof-of concept that the surface of microparticles may be tailored in order to optimise their function as antigen delivery systems and to simultaneously provide an adjuvant effect to induce the immune response. Engineering of microparticles by surface binding of selected, specific ligands to mature and activate DC may increase the effectiveness of microparticulate vaccine delivery systems. Instead of full-size antibodies, we envisage surface grafting of peptide or peptidomimetic ligands.
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